View source for IS Families/IS200-IS605 family-wip

===General===
The IS''200''/IS''605'' family members transpose using obligatory '''s'''ingle '''s'''trand(ss) DNA intermediates<ref name=":22"><pubmed>26104715</pubmed>

</ref> by a mechanism called “'''peel and paste'''”. They differ fundamentally in the organization from classical IS. They have sub-terminal palindromic structures rather than terminal '''IRs'''  ([[:File:Fig. IS200.1.png|Fig. IS200.1]]) and insert 3’ to specific AT-rich tetra- or penta-nucleotides without duplicating the target site.
[[Image:Fig. IS200.1.png|thumb|center|640x640px|'''Fig. IS200.1.''' Genetic organization. '''Left''' (LE) and '''right''' (RE) ends carrying the subterminal hairpin (HP) are presented as red and blue boxes, respectively. Left and right cleavage sites (CL and CR) are presented as black and blue boxes respectively, where the black box also represents element-specific tetra-/pentanucleotide target site (TS). The cleavage positions are indicated by small vertical arrows. Gray arrows: ''tnpA'' and ''tnpB'' open reading frames (orfs); '''(i)''' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group with ''tnpA'' alone; '''(ii)''' to '''(iv)''' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group with ''tnpA'' and ''tnpB'' in different configurations; '''(v)''' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''] group with ''tnpB'' alone.|alt=]]

The transposase, TnpA, is a member of the HUH enzyme superfamily ([[wikipedia:Relaxase|Relaxases]], Rep proteins of RCR plasmids/ss phages, bacterial and eukaryotic transposases of [[IS Families/IS91-ISCR families|IS''91''/IS''CR'' and Helitrons]]<ref><pubmed>26350323</pubmed></ref><ref name=":1"><pubmed>23832240</pubmed>

</ref>)([[:File:Fig. IS200.2.png|Fig. IS200.2]]) which all catalyze cleavage and rejoining of ssDNA substrates. 
[[Image:Fig. IS200.2.png|thumb|center|720x720px|'''Fig. IS200.2.''' The IS''200''/IS''605'' family transposases are “minimal” and the smallest transposases presently know. They include the HUH and Y motifs and use Y as the attacking nucleophile to generate 5’ phosphotyrosine covalent intermediates.  HUH transposases from other transposon families include additional domains.|alt=]]

[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''], the founding member ([[:File:Fig. IS200.3.png|Fig. IS200.3]]), was identified 30 years ago in ''[[wikipedia:Salmonella_enterica_subsp._enterica|Salmonella typhimurium]]''<ref name=":11"><pubmed>6313217</pubmed>
</ref> but there has been renewed interest for these elements since the identification of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group in ''[[wikipedia:Helicobacter_pylori|Helicobacter pylori]]''<ref name=":12"><pubmed>9858724</pubmed>
</ref><ref><pubmed>9631304</pubmed></ref><ref name=":13"><pubmed>11807059</pubmed>
</ref>. Studies of two elements of this group, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] from ''[[wikipedia:Helicobacter_pylori|H. pylori]]'' and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] from the radiation resistant ''[[wikipedia:Deinococcus_radiodurans|Deinococcus radiodurans]]'', have provided a detailed picture of their mobility <ref name=":23"><pubmed>16209952</pubmed>
</ref><ref name=":24"><pubmed>16163392</pubmed>
</ref><ref name=":7"><pubmed>18280236</pubmed></ref><ref name=":0"><pubmed>18243097</pubmed></ref><ref name=":32"><pubmed>20090938</pubmed></ref><ref name=":25"><pubmed>20691900</pubmed>
</ref><ref name=":9"><pubmed>20890269</pubmed></ref>.
[[Image:Fig. IS200.3.png|thumb|center|720x720px|'''Fig. IS200.3.''' '''Top''': [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] Secondary structures in '''LE ('''red) and '''RE''' (blue), promoter (pL), ribosome binding site (RBS), and ''tnpA'' start and stop codons (AUG and UAA) are indicated. '''(i)''' DNA top strand with perfect palindromes at LE and RE in red and blue, interior stem-loop in black, '''(ii)''' RNA stem-loop structure in transcript originated from pL. '''Bottom:''' ''tnpA'' transcription originates at about nt 40, but promoter elements are not defined; the ‘left end’ contains two internal inverted repeats (opposing arrows), one of which acts as a transcription terminator (nts 12–34). The second, (nts 69–138) in the 5’UTR of the tnpA mRNA sequesters the [[wikipedia:Shine-Dalgarno_sequence|Shine-Dalgarno]] sequence. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] in ''[[wikipedia:Salmonella|Salmonella]]'' also expresses a 90 nt sRNA (asRNA, art200, or STnc490) perfectly complementary to the 5’UTR and the first three codons of ''tnpA''. The transcription start site and 3’ end for art200 in ''[[wikipedia:Salmonella|Salmonella]]'' (derived from RNA-Seq experiments) are shown, but promoter elements were not previously defined.|alt=]]

===Distribution and Organization===
The family is widely distributed in prokaryotes with more than 153 distinct members (89 are distributed over 45 genera and 61 species of [[wikipedia:Bacteria|eubacteria]], and 64 are from [[wikipedia:Archaea|archaea]]). It is divided into three major groups based on the presence or absence and on the configuration of two genes: the transposase ''tnpA'', sufficient to promote IS mobility ''in vivo'' and ''in vitro'' and ''tnpB'' of unknown function which is not required for transposition activity ([[:File:Fig. IS200.1.png|Fig. IS200.1]]). These groups are: [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']. TnpB is also present in another [[IS Families/IS607 family|IS family, IS''607'']], which uses a [[wikipedia:Site-specific_recombination|serine-recombinase]] as a transposase. In the phylogeny of this group ([[:File:Fig. IS200.4.png|Fig. IS200.4]]) of IS, both ''tnpB'' and ''tnpA'' of bacterial or archaeal origin are intercalated suggesting some degree of horizontal transfer between these two groups of organisms<ref name=":2"><pubmed>17347521</pubmed>
</ref>.
[[Image:Fig. IS200.4.png|thumb|center|746x746px|'''Fig. IS200.4.''' '''(A)''' Phylogeny-based on ''tnpB'' of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605'']/[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS607 IS''607''] family. '''(B)''' Phylogeny-based on ''tnpA'' of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS607 IS''607''] family (serine recombinase). '''(C)''' Phylogeny-based on ''tnpA'' of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] family (HUH transposase). IS''608'' elements are underlined, single ''orfB'' elements are indicated between brackets, and the asterisk indicates the mosaic construction of the elements of this family (see the text). The various Archaea have been color-coded as follows for clarity: [[wikipedia:Sulfolobales|Sulfolobales]], red; [[wikipedia:Thermoplasmatales|Thermoplasmatales]], magenta; [[wikipedia:Halophile|halophiles]], green; [[wikipedia:Methanogen|methanogens]], blue; “other,” orange. Bacteria are indicated in black.|alt=]]

Isolated copies of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200'']-like ''tnpA'' can be identified in both bacteria and archaea<ref name=":2" />  but, except for a partial copy in ''[[wikipedia:Natronomonas|Natronomonas pharaonis]]'' ([https://www.ncbi.nlm.nih.gov/gene/3702124 NP4630A]), are limited in the archaea to the methanogens ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISMma21 IS''Mma21''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISMba16 IS''Mba16''], and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISMba18 IS''Mba18'']). Full length copies of IS''605''-like elements are also found in bacteria and several archaea and all have corresponding MITEs ('''M'''iniature '''I'''nverted repeat '''T'''ransposable '''E'''lements) derivatives in their host genomes.

====The IS''200'' group====
[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group members encode only ''tnpA'', and are present in gram-positive and gram-negative bacteria and certain archaea<ref><pubmed>10418150</pubmed></ref><ref name=":3"><pubmed>15179601</pubmed>
</ref> ([[:File:Fig. IS200.1.png|Fig. IS200.1]] and [[:File:Fig. IS200.3.png|Fig. IS200.3]]). Alignment of TnpA from various members shows that they are highly conserved but may carry short C-terminal tails of variable length and sequence. 

They can occur in relatively high copy number (e.g. >50 copies of IS''1541'' in ''[[wikipedia:Yersinia_pestis|Yersinia pestis]]'') and are among the smallest known autonomous IS with lengths generally between 600-700 pb. Some members such as [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISW1 IS''W1''] (from ''[[wikipedia:Wolbachia|Wolbachia]]'' sp.) or [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISPrp13 IS''Prp13''] (from ''[[wikipedia:Photobacterium_profundum|Photobacterium profundum]]'') are even shorter. 

[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] was initially identified as an insertion mutation in the ''[[wikipedia:Salmonella_enterica_subsp._enterica|Salmonella typhimurium]]'' histidine operon <ref name=":11" />. It is abundant in different ''[[wikipedia:Salmonella|Salmonella]]'' strains and has now also been identified in a variety of other enterobacteria such as ''[[wikipedia:Escherichia|Escherichia]]'', ''[[wikipedia:Shigella|Shigella]]'' and ''[[wikipedia:Yersinia|Yersinia]]''. 

Different enterobacterial [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] copies have almost identical lengths of between 707 and 711bp. Analysis of the ECOR (''[[wikipedia:Escherichia_coli|E. coli]]'') and SARA (''[[wikipedia:Salmonella|Salmonellae]]'') collections showed that the level of sequence divergence between [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] copies from these hosts is equivalent to that observed for chromosomally encoded genes from the same taxa<ref><pubmed>8253675</pubmed></ref><ref><pubmed>8384142</pubmed></ref>. This suggests that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] was present in the common ancestor of ''[[wikipedia:Escherichia_coli|E. coli]]'' and ''[[wikipedia:Salmonella|Salmonellae]]''.

In spite of their abundance, an enigma of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] behavior is its poor contribution to spontaneous mutation in its original ''[[wikipedia:Salmonella|Salmonella]]'' host: only very rare insertion events have been documented<ref name=":3" />. One reason for these rare insertions could be due to poor expression of the ''tnpA''IS''200'' gene from a weak promoter pL identified at the left IS end (LE)<ref><pubmed>9060429</pubmed></ref><ref name=":15"><pubmed>3009825</pubmed>
</ref> ([[:File:Fig. IS200.3.png|Fig. IS200.3]]).

Besides the characteristic major subterminal palindromes <ref name=":15" /> presumed binding sites of the transposase at both LE and the right end (RE) (Substrate recognition), [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] carries also a potential supplementary interior stem-loop structure ([[:File:Fig. IS200.3.png|Fig. IS200.3]]). These two structures play a role in regulating [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] gene expression. The first (perfect palindrome at LE; nts 12–34) overlaps the ''tnpA''IS''200'' promoter pL, can act as a bi-directional transcription terminator upstream of ''tnpA''IS''200'' and terminates up to 80% of transcripts<ref name=":4"><pubmed>10471738</pubmed></ref> ([[:File:Fig. IS200.3.png|Fig. IS200.3]]). The second (interior stem-loop; nts 69–138) ([[:File:Fig. IS200.3.png|Fig. IS200.3]]), at the RNA level, can repress mRNA translation by sequestration of the [[wikipedia:Ribosome-binding_site|'''R'''ibosome '''B'''inding '''S'''ite (RBS)]] (([[:File:Fig. IS200.3.png|Fig. IS200.3]]). Experimental data suggested that the stem-loop is formed in vivo and its removal by mutagenesis caused up to a 10 fold increase in protein production<ref name=":4" />. Recent deep sequencing analysis revealed another aspect in post-transcriptional regulation of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] expression: A small anti-sense RNA (asRNA) [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] transposase expression (([[:File:Fig. IS200.3.png|Fig. IS200.3]]) was identified as a substrate of [[wikipedia:Hfq_protein|Hfq]], an RNA chaperone involved in post-transcriptional regulation in numerous bacteria<ref><pubmed>18725932</pubmed></ref>. Interestingly, asRNA and [[wikipedia:Hfq_protein|Hfq]] independently inhibit [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] transposase expression: knock-out of both components resulted in a synergistic increase in transposase expression. Moreover, footprint data showed that [[wikipedia:Hfq_protein|Hfq]] binds directly to the 5’ part of the transposase transcript and blocks access to the RBS<ref><pubmed>26044710</pubmed></ref>.

In spite of its very low transposition activity, an increase in [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] copy number was observed during strain storage in stab cultures<ref name=":11" /><ref><pubmed>6315530</pubmed></ref>. However, the factors triggering this activity remain unknown<ref name=":3" />. Transient high transposase expression leading to a burst of transposition was proposed to explain the observed high [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] (>20) copy number in various hosts and in stab cultures <ref name=":11" />.

Although regulatory structures similar to that observed in [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] ([[:File:Fig. IS200.3.png|Fig. IS200.3]]) were predicted in [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1541 IS''1541''], another member of this group with 85% identity to [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''], this element can be detected in higher copy number (> 50) in ''[[wikipedia:Salmonella|Salmonella]]'' and ''[[wikipedia:Yersinia|Yersinia]]'' genomes. However, no detailed analysis of its transposition is available and since no de novo insertions have been experimentally documented and chromosomal copies appear stable in ''[[wikipedia:Yersinia_pestis|Y. pestis]]''<ref><pubmed>9422611</pubmed></ref>, it remains possible that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1541 IS''1541''] also behaves like [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200'']. 

However, the regulatory structures are not systematically present in other [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group members and understanding of the control of transposase synthesis requires further study.

====The IS''605'' group====
[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group members are generally longer (1.6-1.8 kb) due to the presence of a second ''orf'', ''tnpB'' in addition to ''tnpA''. Alignment of TnpA copies from this group indicated that although they do not form a separate clade from the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group TnpA, they generally carry the short C-terminal tail. The ''tnpA'' and ''tnpB'' orfs exhibit various configurations with respect to each other. They may be divergent ([[:File:Fig. IS200.1.png|Fig. IS200.1]] '''i''' top: e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS606 IS''606'']) or expressed in the same direction with ''tnpA'' upstream of ''tnpB''. In these latter cases, the orfs may be partially overlapping ([[:File:Fig. IS200.1.png|Fig. IS200.1]] '''ii''';  e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']) or separate ([[:File:Fig. IS200.1.png|Fig. IS200.1]] '''iii'''; e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISSCpe2 IS''SCpe2''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISEfa4 IS''Efa4'']). ''tnpB'' is also sometimes associated with another transposase, a member of the S-transposases (e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS607 IS''607'']<ref name=":14"><pubmed>10986230</pubmed></ref><ref><pubmed>24195768</pubmed></ref>, see <ref name=":22" />. TnpB was not required for transposition of either [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] or [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''].  

Three related IS, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS606 IS''606''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] ([[:File:Fig. IS200.1.png|Fig. IS200.1]]) have been identified in numerous strains of the gastric pathogen ''[[wikipedia:Helicobacter_pylori|Helicobacter pylori]] <ref name=":12" />''<ref name=":13" /> . [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] is involved in genomic rearrangements in various ''[[wikipedia:Helicobacter_pylori|H. pylori]]'' isolates<ref><pubmed>9789049</pubmed></ref>.

The ''[[wikipedia:Helicobacter_pylori|H. pylori]]'' elements transpose in ''[[wikipedia:Escherichia_coli|E. coli]]'' at detectable frequencies in a standard "mating-out" assay using a derivative of the conjugative [[wikipedia:Fertility_factor_(bacteria)|F plasmid]] as a target <ref name=":12" /> <ref name=":13" />.
 
The two best characterized members of this family are [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and the closely related [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] from ''[[wikipedia:Deinococcus_radiodurans|Deinococcus radiodurans]]''. Both have overlapping ''tnpA'' and ''tnpB'' genes ([[:File:Fig. IS200.1.png|Fig. IS200.1]] '''ii'''). Like other family members, insertion is sequence-specific: [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] inserts in a specific orientation with its left end 3’ to the tetranucleotide TTAC both ''in vivo'' and ''in vitro <ref name=":13" />'' while [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] inserts 3’ to the pentanucleotide TTGAT<ref><pubmed>14676423</pubmed></ref>. Interestingly [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] transposition in its highly radiation resistant Deinococcal host is strongly induced by irradiation<ref name=":26"><pubmed>17006450</pubmed></ref> (Single strand DNA ''in vivo''). Their detailed transposition pathway has been deciphered by a combination of ''in vivo'' studies and ''in vitro'' biochemical and structural approaches ([[IS Families/IS200-IS605 family#Mechanism of IS200.2FIS605 single strand DNA transposition|Mechanism of IS''200''/IS''605'' single strand DNA transposition]]).

====The IS''1341'' group====
Elements of the third group, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''], are devoid of ''tnpA'' and carry only ''tnpB'' ([[:File:Fig. IS200.1.png|Fig. IS200.1]] '''v'''). The IS occurs in three copies in [[wikipedia:Thermophile|Thermophilic bacterium]] PS3<ref><pubmed>7557457</pubmed></ref>. Multiple presumed full-length elements (including ''tnpA'' and ''tnpB'') and closely related copies have been identified in other bacteria such as ''[[wikipedia:Geobacillus|Geobacillus]]''. On the other hand, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS891 IS''891''] from the cyanobacterium ''[[wikipedia:Anabaena|Anabaena]]'' is present in multiple copies on the chromosome and is thought to be mobile since a copy was observed to have inserted into a plasmid introduced in the strain<ref name=":33"><pubmed>2553665</pubmed></ref>.

Another isolated ''tnpB''-related gene, ''[https://www.uniprot.org/uniprot/Q50HS5 gipA]'', present in the ''[[wikipedia:Salmonella|Salmonella]]'' Gifsy-1 prophage may be a virulence factor since a ''gipA'' null mutation compromised ''[[wikipedia:Salmonella|Salmonella]]'' survival in a Peyer's patch assay<ref><pubmed>10913072</pubmed></ref>. While no mobility function has been suggested for ''gipA'', it is indeed bordered by structures characteristic of IS''200''/IS''605'' family ends and closely related to ''[[wikipedia:Escherichia_coli|E. coli]]'' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISEc42 IS''Ec42''].

In spite of their presence in multiple copies, it is still unclear whether [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''] group members are autonomous IS or products of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group degradation and require TnpA supplied from a related IS in the same cell for transposition. 

====IS decay====
Circumstantial evidence based on analysis of the [https://isfinder.biotoul.fr/ ISfinder database] suggests that IS carrying both ''tnpA'' and ''tnpB'' genes may be unstable. Thus, although members of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group are often present in high copy number in their host genomes, intact full-length [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group members are invariably found in low copy number ([https://scholar.google.com/citations?user=WHAtfqcAAAAJ&hl=pt-BR P. Siguier], unpublished) (See also [[IS Families/IS200-IS605 family#TnpB|TnpB]]). On the other hand, various truncated [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group derivatives appear quite frequently.  

These forms seem to result from successive internal deletions and retain intact LE and RE copies. Sometimes, as in the case of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISSoc3 IS''Soc3''], orf inactivation appears to have occurred by successive insertion/deletion of short sequences (indels) generating frameshifts and truncated proteins. For some IS (e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISCco1 IS''Cco1''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISTel2 IS''Tel2''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISCysp14 IS''Cysp14''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISSoc3 IS''Soc3'']) degradation can be precisely reconstituted and each successive step validated by the presence of several identical copies ([https://scholar.google.com/citations?user=WHAtfqcAAAAJ&hl=pt-BR P. Siguier], unpublished). This suggests that the degradation process is recent and that these derivatives are likely mobilized by TnpA supplied in trans by autonomous copies in the genome. 

===Mechanism of IS''200''/IS''605'' single strand DNA transposition===

====Early models====
A number of alternative mechanisms were initially proposed to explain [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISHp608 IS''608''] transposition <ref name=":24" /> ([[:File:FigIS200-4b.png|Fig. IS200.4b]]). These all included the insertion of a double-strand circular transposon copy ([[:File:FigIS200-4b.png|Fig. IS200.4b]] '''D'''). One model ([[:File:FigIS200-4b.png|Fig. IS200.4b]] '''A''') envisaged simultaneous or consecutive cleavage at '''LE''' and '''RE''' and reciprocal strand transfer would generate a [[wikipedia:Holliday_junction|'''H'''olliday '''j'''unction (HJ)]] which then could be resolved into double-strand circular copies of the transposon. The second ([[:File:FigIS200-4b.png|Fig. IS200.4b]] '''B''') cleavage at '''LE''' and replicative strand displacement using a 3’OH of the flanking donor DNA. This could assist formation of a single strand region accessible for cleavage of '''RE''' to generate a single-strand transposon circle which could be replicated into a double-strand copy. The third ([[:File:FigIS200-4b.png|Fig. IS200.4b]] '''C''') proposed cleavage at '''LE''' with displacement of the transposon strand to form a single strand loop. Subsequent ''in vitro'' and ''in vivo'' experiments (below) demonstrated that not only was [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISHp608 IS''608''] capable of excision as a single-strand DNA circle but that this could be inserted into a single strand target.
[[File:FigIS200-4b.png|center|thumb|720x720px|'''Fig. IS200.4b.''' Proposed Models for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] Transposition. Donor and target replicons are indicated (full and dotted lines, respectively). Dashed lines indicate newly replicated DNA. The conserved target sequence '''TTAC''' is also indicated. '''A)''' simultaneous or consecutive cleavage at '''LE''' and '''RE''' and reciprocal strand transfer would generate a [[wikipedia:Holliday_junction|'''H'''olliday '''j'''unction (HJ)]] which could be resolved into double-strand circular copies of the transposon; '''B)''' Cleavage at '''LE''' and replicated strand displacement using a 3’OH of the flanking donor DNA. This could assist the formation of a single strand circle region accessible for cleavage of '''RE''' to generate a single-strand transposon circle which could be replicated into a double-strand copy. '''C)''' Cleavage at '''LE''' with a displacement of the transposon strand to form a single strand loop. '''D)''' Integration. From Ton-Hoang et al.<ref name=":24" />.]]
<br />
====General transposition pathway====
The transposition pathway of IS''200''/IS''605'' family members is shown in [[:File:Fig. IS200.5.png|Fig. IS200.5]]. Much of the biochemistry was elucidated using an [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] cell-free ''in vitro'' system which recapitulates each step of the reaction. This requires purified TnpAIS''608'' protein, single strand [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] DNA substrates and divalent metal ions such as Mg2+ or Mn2+<ref name=":24" /><ref name=":7" /><ref name=":0" />. Similar and complementary results were also obtained with [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']<ref><pubmed>20090938</pubmed></ref><ref name=":25" /><ref name=":9" />. The reactions are not only strictly dependent on single strand (ss) DNA substrates but are also strand-specific: only the “top” strand (defined as the strand carrying target sequence, TS, 5’ to the IS; [[:File:Fig. IS200.5.png|Fig. IS200.5]] '''top''') is recognized and processed whereas the “bottom” strand is refractory <ref name=":24" /> <ref name=":7" />. Cleavage of the top strand at the left and right cleavage sites (TS/CL and CR, note that TS is also the left cleavage site CL) ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''B''') leads to excision as a circular ssDNA intermediate with abutted left and right ends (transposon joint) ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''C''' bottom left). This is accompanied by rejoining of the DNA originally flanking the excised strand (donor joint).
[[Image:Fig. IS200.5.png|thumb|center|720x720px|'''Fig. IS200.5.''' '''Top''': [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] organization. The left (LE) and right (RE) ends with a subterminal hairpin (HP) are in red and blue, left and right cleavage sites (CL/TS and CR) are represented by black and blue boxes, respectively. Bottom left: Excision. '''(A)''' TnpA activity: top strand (active strand) structures are recognized and cleaved by TnpA (vertical arrows). '''(B)''' Upon cleavage, a 5′ phosphotyrosine bond (green cylinder) is formed with LE, and with the RE 3′ flank and 3′-OH (yellow circle) is formed at the left flank and RE. '''(C)''' Excision of the IS''608'' single-strand circle intermediate with abutted LE and RE (RE–LE junction or transposon joint) accompanied by the formation of donor joint retaining the target sequence. '''Bottom right''': Integration.
'''(D)''' Transposon circle with the transposon joint and target DNA (black) with the target site. '''(E)''' TnpA catalyzes the cleavage of transposon joint and single-strand target. '''(F)''' Integration.|alt=]]

The transposon joint is then cleaved ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''E''' bottom right) and integrated into a single strand conserved element-specific target sequence (TS) where the left end invariably inserts 3’ to TS ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''F'''). This target specificity is another unusual feature of IS''200''/IS''605'' transposition. The target sequence is characteristic of the particular family member and, although it is not part of the IS, it is essential for further transposition because it is also the left end cleavage site CL of the inserted IS <ref name=":24" /> (The Single strand Transpososome and Cleavage site recognition) and is therefore intimately involved in the transposition mechanism. 

====TnpA, Y1 transposases and transposition chemistry====
IS''200''/IS''605'' family transposases belong to the '''HUH enzyme superfamily.''' All contain a conserved amino-acid triad composed of Histidine (H)-bulky hydrophobic residue (U)-Histidine (H)<ref><pubmed>8374079</pubmed></ref> providing two of three ligands required for coordination of a divalent metal ion that localizes and prepares the scissile phosphate for nucleophilic attack. HUH proteins catalyze ssDNA breakage and joining with a unique mechanism. They all catalyse DNA strand cleavage using a transitory covalent 5' phosphotyrosine enzyme-substrate intermediate and release a 3' OH group<ref name=":1" /> ([[:File:1.41.1.png|Fig. 23.4]]). 

The HUH enzyme family also includes other transposases of the [[IS Families/IS91-ISCR families|IS''91''/IS''CR'' and Helitron families]] as well as proteins involved in DNA transactions essential for plasmid/virus rolling circle replication (Rep; not to be confused with the TnpAREP/REP system described in [[IS Families/IS200-IS605 family#Y1 transposase domestication|Domestication]]) and plasmid conjugation (Mob/relaxase) ([[General Information/Major Groups are Defined by the Type of Transposase They Use#Groups with HUH Enzymes|Groups with HUH Enzymes]]; [[:File:1.10.1.png|Fig.7.5]]). 

IS''200''/IS''605'' transposases are single-domain proteins containing a single catalytic tyrosine residue, called '''Y1 transposase'''. They use the tyrosine residue (Y127 for IS608) as a nucleophile to attack the phosphodiester link at the cleavage sites (vertical arrows in [[:File:Fig. IS200.5.png|Fig. IS200.5]] '''A''' and '''D'''). Since cleavages at both IS ends occur on the same strand, the polarity of the reaction implies that the enzyme forms a covalent 5’-phosphotyrosine bond with the IS at LE producing a 3’-OH on the DNA flank and a 5’-phosphotyrosine bond at the RE flank producing a 3’-OH on RE itself ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''B'''). The released 3′-OH groups then act as nucleophiles to attack the appropriate phospho-tyrosine bond resealing the DNA backbone in one case and generating a single-strand DNA transposon circle in the other ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''C'''). The same polarity is applied to the integration step ([[:File:Fig. IS200.5.png|Fig. IS200.5]] '''D''', '''E''' and '''F'''). As an important mechanistic consequence of this chemistry, IS''200''/IS''605'' transposition occurs without loss or gain of nucleotides. ''In vitro'', the reaction requires only TnpA and does not require host cell factors. 

====TnpA overall structure====
Crystal structures of Y1 transposases have been determined for three family members: [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] (TnpAIS608) from ''[[wikipedia:Helicobacter_pylori|Helicobacter pylori]]'' <ref name=":23" /><ref name=":0" />, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] (TnpAISDra2) from ''[[wikipedia:Deinococcus_radiodurans|Deinococcus radiodurans]]<ref name=":9" />'' and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISC1474 IS''C1474''] from ''[[wikipedia:Sulfolobus_solfataricus|Sulfolobus solfataricus]]''<ref name=":16"><pubmed>16340015</pubmed></ref>. In contrast to most characterised HUH enzymes, which are usually monomeric and have two catalytic tyrosines, Y1 transposases form obligatory dimers with two active sites ([[:File:Fig. IS200.6.png|Fig. IS200.6]] '''A'''). The two monomers dimerize by merging their β-sheets into one large central β-sheet sandwiched between α-helices. Each catalytic site is constituted by the HUH motif from one TnpA monomer (H64 and H66 in the case of TnpAIS608) and a catalytic tyrosine residue (Y127) located in the C-terminal αD helix tail of the other monomer ([[:File:Fig. IS200.6.png|Fig. IS200.6]] '''A'''). This is joined to the body of the protein by a flexible loop (trans configuration, Active site assembly and Catalytic activation and [[IS Families/IS200-IS605 family#Transposition cycle: the trans.2Fcis rotational model|Transposition cycle: the trans/cis rotational model]]).
[[Image:Fig. IS200.6.png|thumb|center|600x600px|'''Fig. IS200.6.''' '''(A)''' Crystallographic structure of TnpA alone. The two monomers of the TnpA dimer are colored green and orange, respectively. Positions of helix αD and catalytic residues are shown. '''(B)''' Co structure TnpA–RE HP22. HP22 is shown in blue. The extrahelical T17 and the T located in the hairpin loop are indicated in red (6). Note that in the TnpA–HP22 co-structure, binding sites for the hairpins are located on the same face of the TnpA dimer whereas the two catalytic sites are formed on the opposite surface (A, C–F).|alt=]]

The TnpA enzyme active sites are believed to adopt two functionally important conformations: the trans configuration described above ([[:File:Fig. IS200.6.png|Fig. IS200.6]] '''A'''), in which each active site is composed of the HUH motif supplied by one monomer with the tyrosine residue supplied by the other, and the cis configuration, in which both motifs are contributed by the same monomer (IS''200''/IS''605'' '''video 1''' '''below'''; kindly supplied by [https://www.embl.de/research/units/scb/barabas/ O. Barabas] and [https://www-mslmb.niddk.nih.gov/dyda/dydalab.html Fred Dyda]). The trans conformation is active during cleavage where Tyrosine acts as nucleophile whereas the cis conformation is thought to function during strand transfer where the 3’OH is the attacking nucleophile ([[IS Families/IS200-IS605 family#Transposition cycle: the trans.2Fcis rotational model|Transposition cycle: the trans/cis rotational model]]). Only the trans configuration of TnpAIS''608'' and TnpAIS''Dra2'' has yet been observed crystallographically <ref name=":23" /><ref name=":9" /> but the existence of the cis configuration is supported by biochemical data <ref name=":8"><pubmed>23345619</pubmed></ref>.
<center>
{| class="wikitable"
![[File:IS200 S605-video-1.mp4|center|380x380px]]'''<small>IS''200''/IS''605'' video 1</small>'''
|}
</center>

====The Single strand Transpososome====
The key machinery for transposition is the higher-order protein-DNA complex, the transpososome (or synaptic complex) which contains both transposase and two IS DNA ends with or without target DNA. Transpososome formation, stability, and the temporal changes in a configuration which occur during the transposition cycle have been characterized for TnpAIS''608'' by crystallographic and biochemical approaches. Although for technical reasons it was not possible to obtain structures with both LE and RE hairpins together, co-crystal structures with either LE or RE showed that a TnpA dimer binds two subterminal DNA hairpins suggesting that it could bind both LE and RE ends simultaneously. Binding sites for the hairpins are located on the same face of the TnpA dimer while the two catalytic sites are formed on the opposite surface ([[:File:Fig. IS200.6.png|Fig. IS200.6]] '''A''' and '''B)''' (IS''200''/IS''605'' '''video 2 below'''; kindly supplied by [https://www.embl.de/research/units/scb/barabas/ O. Barabas] and [https://www-mslmb.niddk.nih.gov/dyda/dydalab.html Fred Dyda]). The hairpin forms a distorted helix anchored by base interactions at the foot (IS''200''/IS''605'' '''video 2 below'''; kindly supplied by [https://www.embl.de/research/units/scb/barabas/ O. Barabas] and [https://www-mslmb.niddk.nih.gov/dyda/dydalab.html Fred Dyda]).
<br />
<center>
{| class="wikitable"
![[File:IS200 S605-video-2 1.mp4|center|380x380px]]'''<small>IS''200''/IS''605'' video 2</small>'''
|}</center><br />

=====Substrate recognition=====
A key feature of TnpA is that it is only active on one strand, the “top” strand. The [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] ends carry subterminal imperfect hairpins. In addition to specific sequences on the loops, the irregularities on the hairpins help the enzyme to distinguish between “top” and “bottom” strands <ref name=":23" /> <ref name=":9" />. The initial co-crystal structure was obtained with TnpAIS''608'' and a 22nt imperfect RE hairpin (HP22) including its characteristic extrahelical T17 located mid-way along the DNA stem ([[:File:Fig. IS200.6.png|Fig. IS200.6]] and [[:File:Fig. IS200.7.png|Fig. IS200.7]]). In addition to a number of backbone contacts with HP22, TnpAIS''608'' also shows several base-specific contacts, in particular with T10 in the loop and the extrahelical T17<ref name=":23" /> ([[:File:Fig. IS200.6.png|Fig. IS200.6]] '''B'''). Exchange of T10 and neighboring T nucleotides in the loop abolished binding whereas the exchange of T17 for an A significantly reduced but did not eliminate binding <ref name=":10"><pubmed>21745812</pubmed></ref>. Similar studies with TnpAIS''Dra2'' showed that it also recognises a similarly located T in the hairpin loop of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] and that this is essential for binding <ref name=":9" /> . Instead of an extrahelical T, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] LE and RE include a bulge caused by two mismatched nucleotides (G and T) in the hairpin stem. These unpaired nucleotides are specifically recognized and stabilized by the protein. Again, mutation of the T (to C which, in this case, eliminates the bulge to generate a GC base pair in the stem) greatly reduces binding (IS''200''/IS''605'' '''video 3A below'''; kindly supplied by [https://www.embl.de/research/units/scb/barabas/ O.Barabas] and [https://www-mslmb.niddk.nih.gov/dyda/dydalab.html Fred Dyda]). 

Although most members of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] group, which includes [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''], have imperfect palindromes with extrahelical bases or bulges, some members of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group (e.g [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1541 IS''1541'']) include perfect hairpins. Whether base-specific interactions with the loop sequence is exclusively responsible for strand-specific activity of the corresponding transposase remains to be clarified.<center>
{| class="wikitable"
![[File:IS200 S605-video-3A.mp4|center|380x380px]]<small>IS''200''/IS''605'' '''video 3A'''</small>
|}</center>
=====Cleavage site recognition=====
The left (CL/TS) and right (CR) [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] cleavage sites (TTACl and TCAAl respectively, where l represents the point of cleavage) are located some distance from the subterminal recognition hairpins (19 nt at LE and 10 nt at RE) ([[:File:Fig. IS200.7.png|Fig. IS200.7]]). The system is asymmetric because the two distinct cleavage sites are separated from the hairpins by linkers of different lengths and the CL/TS sequence does not form part of IS while CR does.
[[Image:Fig. IS200.7.png|thumb|center|680x680px|'''Fig. IS200.7.''' Canonical and noncanonical base interactions in '''(A)''' '''left end''' (LE) and '''(B)''' '''right end''' (RE). LE and RE (red and blue). Cleavage sequences CL or CR (black or dark blue boxes); guide sequences GL and GR pink or light blue, respectively. Two nucleotides at the 3′ foot of HPL, R involved in triplet formation are highlighted by bold and in a black frame. LE and RE and the base paring within HPL and HPR are shown. Insets show interactions between cleavage and guide sequences. Filled lines: canonical base interactions, dotted lines: additional noncanonical base interactions.|alt=]]

Structural studies revealed that the cleavage sites are recognized in a unique way that does not involve direct sequence recognition by TnpA. Instead, an internal part of the IS sequence is co-opted to recognize different cleavage sites allowing TnpA to catalyze both excision and integration of the element with a single DNA binding domain.

Internal transposon sequences, the left (GL) and right (GR) tetranucleotide guide sequences, AAAG and GAAT, located 5’ to the foot of the hairpins ([[:File:Fig. IS200.7.png|Fig. IS200.7]]), recognize their respective cleavage sites by direct base interactions. These GL/CL and GR/CR interactions involve 3 of the 4 nt of GL and GR. They include both [[wikipedia:Base_pair|canonical Watson-Crick interactions]] and in the case of RE, non-canonical interactions resulting in base triplets ([[:File:Fig. IS200.7.png|Fig. IS200.7]] and [[:File:Fig. IS200.8.png|Fig. IS200.8]], bases joined by both regular and dotted lines respectively). In the case of LE and the transposon joint, base triples (dotted lines) are suggested from biochemical data <ref name=":10" /> (IS''200''/IS''605'' '''video 3B below'''; kindly supplied by [https://www.embl.de/research/units/scb/barabas/ O. Barabas] and [https://www-mslmb.niddk.nih.gov/dyda/dydalab.html Fred Dyda]). 
[[Image:Fig. IS200.8.png|thumb|center|600x600px|'''Fig. IS200.8.''' Structure of the co-complex TnpAIS608–RE35 adapted from reference 8 showing the active site and the base pairs between CR (TCAA, dark blue) and GR (GAAT, light blue). The gray sphere is bound Mn2+. '''Right''': Two base triplets observed in the TnpAIS608–RE35 complex.|alt=]]

<center>
{| class="wikitable"
![[File:IS200 S605-video-3B.mp4|center|381x381px]]<small>IS''200''/IS''605'' '''video 3B'''</small>
|}
</center>These interactions place the scissile phosphate precisely into the two active sites of TnpAIS''608'' for nucleophilic attack by the catalytic Y127. Interestingly, the base-pairing patterns responsible for cleavage site recognition are similar at LE, RE and the target site in spite of sequence differences ([[:File:Fig. IS200.7.png|Fig. IS200.7]], [[:File:Fig. IS200.8.png|Fig. IS200.8]], [[:File:Fig. IS200.9.png|Fig. IS200.9]]). Since TS is also CL, this type of recognition not only explains the requirement for the TS located at the left end of the inserted IS ([[:File:Fig. IS200.5.png|Fig. IS200.5]], [[:File:Fig. IS200.9.png|Fig. IS200.9]]) for further transposition, but also the target specificity. Upon integration, TS is presumably recognized by the GL present on the excised transposon joint. Note that the transposon joint contains only the LE guide sequence GL but not the LE cleavage site CL ([[:File:Fig. IS200.5.png|Fig. IS200.5]], [[:File:Fig. IS200.9.png|Fig. IS200.9]]).
[[Image:Fig. IS200.9.png|thumb|center|680x680px|'''Fig. IS200.9.''' Target recognition: single-strand transposon joint (RE–LE junction) and target Ts are presented. For simplicity, only the recognition of the target cleavage site is indicated. LE and RE are shown in red and blue. Cleavage sequences C<sub>L</sub> or C<sub>R</sub> are placed in black or dark blue boxes; guide sequences G<sub>L</sub> and G<sub>R</sub> are framed in pink and light blue, respectively. Two nucleotides at the 3′ foot of  the left and right hairpin structures HP<sub>L</sub> and HP<sub>R</sub> involved in triplet formation are highlighted by bold and are in a black

frame. Nucleotide sequences of LE and RE and the base paring within HP<sub>L</sub> and HP<sub>R</sub> are shown. The inset figures describe the interactions between the cleavage sequences and guide sequences. The filled lines indicate canonical base interactions and the dotted lines indicate additional noncanonical base interactions.|alt=]]

Similar crystal structures were obtained with TnpAIS''Dra2'' (see also Single strand DNA ''in vivo'') with a similar interaction network between the guide sequences and cleavage sites. The [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] transpososome is structurally very similar to those of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] despite only 34% sequence identity of the transposases. It is important to note that the target sequence in [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] is a pentanucleotide instead of a tetranucleotide as in [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608'']. The fifth nucleotide in the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] sequence is however not involved in DNA-DNA interactions but in DNA-protein interaction <ref name=":9" />.

The potential cleavage site recognition mode (i.e. the canonical interaction network between CL,R and GL,R) is indeed well conserved throughout the family ([[:File:Fig. IS200.10.png|Fig. IS200.10]]).
[[Image:Fig. IS200.10.png|thumb|center|720x720px|'''Fig. IS200.10.''' Multiple sequence alignment of the cleavage sites and guide sequences using [http://weblogo.berkeley.edu/ Weblogo] was carried out on 38, 43 and 23 members of the IS''200'' '''(i)''', the IS''605'' '''(ii)''', and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''] '''(iii)''' groups, respectively.|alt=]]

This model has been validated ''in vitro'' and ''in vivo'' by showing that it is possible to modify cleavage sites by changing corresponding guide sequences. Moreover, in the case of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''], modifications of GL in the transposon joint generate predictable changes in insertion site-specificity of the element<ref name=":5"><pubmed>19524540</pubmed></ref>.

The [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] recognition system has also been modified to include additional sequences which assist more specific targeting of insertions<ref><pubmed>29635476</pubmed></ref>.

=====Active site assembly and Catalytic activation=====
Comparison of crystal structures of different TnpA protein-DNA complexes<ref name=":23" /><ref name=":0" /> <ref name=":16" /> revealed TnpA in both active and inactive configurations. In both the free TnpAIS''608'' dimer and TnpAIS''608''-DNA complexes bound to a “minimal” HP22 hairpin (which does not include the guide sequence), the catalytic tyrosine residue (Y127) points away from the HUH motif (H64 and H66) and therefore cannot act as a nucleophile <ref name=":23" /> ([[:File:Fig. IS200.11.png|Fig. IS200.11]]). 

The enzyme is therefore in an inactive conformation. Binding to the appropriate substrate containing the 4 nucleotide guide sequence 5’ to the hairpin foot (compare [[:File:Fig. IS200.11.png|Fig. IS200.11]] '''left''' and '''right''') triggers a change in TnpA configuration that permits assembly of functional active sites. A single A (A+18, [[:File:Fig. IS200.7.png|Fig. IS200.7]] and [[:File:Fig. IS200.8.png|Fig. IS200.8]]) in the guide sequence present in both GL and GR does not participate in base interactions with the cleavage site. On formation of the CL(R)/GL(R) base interaction network, this single base penetrates the structure and forces the C-terminal αD helix carrying Y127 closer to the HuH motif placing it in the correct position poised for catalysis <ref name=":0" /> (compare [[:File:Fig. IS200.11.png|Fig. IS200.11]] '''left''' and '''right'''; [[:File:Fig. IS200.12.png|Fig. IS200.12]])(IS''200''/IS''605'' '''video 4 below'''; kindly supplied by [https://www.embl.de/research/units/scb/barabas/ O. Barabas] and [https://www-mslmb.niddk.nih.gov/dyda/dydalab.html Fred Dyda]). 

This movement also places a third amino acid (Q131 located at the C-terminal end of helix αD on the same face as Y127) in a position enabling it to function in conjunction with both H residues to complete the metal ion binding pocket. This movement is made possible by the fact that the αD helix is attached to the protein body by a flexible loop. This conformational change involving αD helix movement will be discussed below ([[IS Families/IS200-IS605 family#Transposition cycle: the trans.2Fcis rotational model|Transposition cycle: the trans/cis rotational model]]).
[[Image:Fig. IS200.11.png|thumb|center|680x680px|'''Fig. IS200.11''' The presence of the guide sequence AAAG at the foot of IPL results in the movement of helices αD and places 
tyrosine Y127 in the correct position with respect to the HUH to form the active site.|alt=]]
[[Image:Fig. IS200.12.png|thumb|center|500x500px|'''Fig. IS200.12.''' '''(C)''' Configuration of the active site in the TnpA–RE HP22. HP22 is shown in blue. Note that in A, B and C, TnpA is in the inactive conformation. The arrow shows the presumed rotation of the αD helix to activate the protein. '''(D)''' Configuration of the active site in the TnpA–LE HP26 costructure. LE HP26 is shown in red and the 5′ 4-nucleotide extension (GL) in yellow). The base A+18 has displaced Y127 to activate the protein. (Adapted from references 6 and 8.)|alt=]]<br />

<center>
{| class="wikitable"
![[File:IS200 S605-video-4.mp4|center|381x381px]]<small>IS''200''/IS''605'' '''video 4'''</small>
|}</center><br />

=====Transpososome assembly and stability=====
Excision requires the assembly of a transpososome containing both LE and RE. However, it is technically difficult to generate crystallographically pure complexes of this type. Only crystal structures containing two LE or two RE were obtained. The excision transpososome was initially modelled using information obtained from the IS608LE-TnpA and RE-TnpA structures<ref name=":0" /> ([[:File:Fig. IS200.6.png|Fig. IS200.6]] '''B'''; [[:File:Fig. IS200.13.png|Fig. IS200.13]]). However, complexes containing both LE and RE have now been identified using a band shift assay and characterized biochemically<ref name=":10" />.
[[Image:Fig. IS200.13.png|thumb|center|600x600px|'''Fig. IS200.13.''' '''(E)''' TnpA–RE35 complex. Interaction of GR-CR (in light and dark blue, respectively) positions the cleavage site within the catalytic site of the protein. '''(F)''' Modeled TnpA–LE–RE complex. LE, RE, and flanking sequences in red, blue, and black, respectively. |alt=]]

A TnpA co-complex with either LE or RE can be titrated by the addition of increasing quantities of the other end (RE or LE) to obtain a transpososome containing both LE and RE. This can be easily detected in a gel shift assay. Such species proved to be catalytically active since they could be removed from the gel and, when incubated with the essential divalent metal ion, robust reaction products could be detected in a denaturing ge<ref name=":10" />.

This approach was used to monitor both transpososome formation and stability using oligonucleotides carrying point mutations in GL,R and CL,R. Robust transpososome formation and cleavage activity requires much of the network of GL,R and CL,R interactions observed in the crystal structures<ref name=":10" /> (schematised in [[:File:Fig. IS200.7.png|Fig. IS200.7]]). Although base triplets in the original LE co-crystal structure were not detected since the LE substrate was too short<ref name=":0" />, the biochemical data suggested that such interactions probably exist (grey dotted lines in [[:File:Fig. IS200.7.png|Fig. IS200.7]]). For example, the two nucleotides 3’ to the foot of the LE hairpin (at equivalent positions to triplet forming bases in RE, [[:File:Fig. IS200.7.png|Fig. IS200.7]]  are required for robust synaptic complex formation and cleavage<ref name=":10" />. This further implies that these base triplets might also be involved in target DNA capture (grey dotted lines in [[:File:Fig. IS200.9.png|Fig. IS200.9]]). 

Base changes in GL resulted in a predictable choice of target sequence<ref name=":5" />. However, large differences in insertion frequencies were observed. The influence of the presumed non-canonical interactions in LE would provide an explanation for this variability since these were not taken into account in the choice of LE guide sequence.

In both [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''], the extra-helical bases in the hairpin stem and nucleotides in the loop are also important for transpososome formation even in a context which includes both GL,R and CL,R <ref name=":9" /><ref name=":10" />.

====Transposition cycle: the trans/cis rotational model====
Transpososome assembly is followed by two critical chemical steps: cleavage and strand transfer. These are thought to be accomplished by a series of large changes in transpososome configuration. A detailed model has been proposed for the dynamics of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] transpososome during the transposition reactions<ref name=":0" /><ref name=":8" /> ([[:File:Fig. IS200.13.png|Fig. IS200.13]]; IS''200''/IS''605'' '''video 1'''). As described in TnpA overall structure (above), TnpAIS''608'' could in principle assume two configurations: trans and cis. Switching between these two states would involve rotation of the two unconstrained flexible arms which join the αD helix to the protein body. 

The current model for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] transposition proposes that the strand transfer step involves rotation of these arms from the trans to the cis configuration: cleavage occurs while the enzyme is in the trans configuration. A trans to cis conformational change then occurs allowing strand transfer. The ground state of the IS''608'' and IS''Dra2'' transpososomes obtained from crystallography is the trans configuration. LE and RE binding and cleavage occur with the enzyme in its trans configuration ([[:File:Fig. IS200.13.png|Fig. IS200.13]]; IS''200''/IS''605'' '''video 1'''). 

This results in the formation of the 5’ phosphotyrosine bond with LE liberating a 3’-OH on the flanking DNA and the 5’phosphotyrosine bond with the RE DNA flank liberating a 3’-OH on the RE transposon end. Rotation of the two arms would displace LE towards the sequestered 3’-OH of RE and the RE flank towards the 3’-OH of the LE flank ([[:File:Fig. IS200.7.png|Fig. IS200.13]]; IS''200''/IS''605'' '''video 1''') and position them so that both 3’-OH can attack the appropriate phosphodiester bond. This model is supported by several lines of indirect evidence from studies of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''].

An initial piece of evidence concerns the length differences in the LE and RE “linker” (the distance between the hairpin foot and the cleavage site): this is only 10 nt for RE but 19 nt for LE ([[:File:Fig. IS200.9.png|Fig. IS200.9]]). The rotation model suggests that the longer LE linker may be required to provide sufficient length to rotate the 5’ LE phospho-tyrosine bond to position it closes the immobile RE 3’-OH ([[:File:Fig. IS200.13.png|Fig. IS200.13]]; IS''200''/IS''605'' '''video 1'''). This would imply that LE linker length is critical for strand transfer. Indeed, sequential reduction in the length of the LE linker has a large effect on transposition frequency and excision ''in vivo''. ''In vitro'', it also had a somewhat larger effect on strand transfer than on cleavage <ref name=":10" />, supporting the idea that the linker is important for mechanical movement. However, transpososome formation and stability was also observed to be affected with the shortest linkers. This presumably reflects steric barriers to GL(R)/CL(R) interaction and supports the notion that these interactions are important in transpososome assembly.  A survey of over 100 different IS from all three groups (35 from the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group; 47 from [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] and 24 from [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']) in the public databases has shown that the asymmetry of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] ends is conserved across the entire family: the left linker is always longer than the right (15-16 nt versus 8 nt)<ref name=":8" /> ([[:File:Fig. IS200.14.png|Fig. IS200.14]]).
[[Image:Fig. IS200.14.png|thumb|center|640x640px|'''Fig. IS200.14.''' Linker length distribution of LE and RE from 76 (red) and 80 (blue) different IS, respectively.|alt=]]

The second piece of evidence comes from the behaviour of TnpAIS''608'' [[wikipedia:Protein_dimer|heterodimers]] carrying point mutations in the HuH or catalytic Y. These were expressed and assembled ''in vivo'' and purified based on two different C-terminal affinity tags (one for each monomer). This permitted [[wikipedia:Protein_dimer|heterodimers]] to be distinguished form [[wikipedia:Protein_dimer|homodimers]]. A [[wikipedia:Protein_dimer|heterodimer]] with a combination of mutations that enforce a trans-active TnpA site (in which the wildtype HuH motif and Y127 belong to different TnpA monomers)  is proficient for cleavage but not for rejoining. In contrast, a heterodimer with cis-active TnpA site (in which the wildtype HuH motif and Y127 belong to the same TnpA monomer) is proficient for rejoining but inactive in cleavage<ref name=":8" />. This implies that all chemical reactions involved in cleavage occur in the trans site while the chemical reactions for strand transfer occur in the cis site. This strongly supports the rotational model. 

A third piece of evidence comes from studies of the flexible arm that joins helix αD to the body of the protein and which is proposed to play a pivotal role in the rotation. This flexibility may be facilitated by two glycine residues (G117 and G118). Mutation of these two residues did not affect strand cleavage but led to inhibition of strand transfer suggesting that the two residues are required for achieving a cis configuration. The importance of these G residues is reflected in their conservation throughout the family<ref name=":8" />.

Thus, while the cis configuration has not been observed crystallographically for these elements, its existence is strongly suggested by experimental data, supporting the trans/cis rotational model ([[:File:Fig. IS200.15.png|Fig. IS200.15]]).
[[Image:Fig. IS200.15.png|thumb|center|740x740px|'''Fig. IS200.15.''' Strand transfer and reset model of IS''608'' transpososome. '''(A)''' The inactive form of TnpA dimer in the absence of DNA (pale green, orange ovals, and dark green and orange cylinders represent the body and the αD helices of two monomers, respectively). At the ends, dotted red and blue lines represent linkers at the left end ('''LE''') and the right end ('''RE'''), light red and light blue boxes represent GL and GR, respectively. '''(B)''' Binding of a copy of LE and RE resulting in TnpA activation (catalytic sites in trans). '''(C)''' Cleavage of both ends forms a 5′ phosphotyrosine linkage between Y127 and '''LE''' on one αD helix (dark orange cylinders) and between Y127 and the RE flank on the other (dark green cylinders). 3′-OH groups are shown as yellow circles. Reciprocal rotation of both αD helices from trans to the cis configuration is indicated by large arrows. '''(D)''' Strand transfer takes place to reconstitute the joined donor backbone (donor's joint) and generate the '''RE'''–'''LE''' transposon junction at cis configuration. '''(E)''' Release of the donor's joint and transition from cis to trans configuration. '''(F)''' Reset to the transform and target site engagement. '''(G)''' Cleavage of the '''RE'''–'''LE''' junction and target and transition from trans to cis configuration. '''(H)''' Regeneration of the left and right transposon ends.|alt=]]<br />

<br />
===Regulation of single strand transposition===
====Single strand DNA ''in vivo''====
The obligatory single-stranded nature of IS''200''/IS''605'' transposition ''in vitro'' suggests that it is limited in vivo by the availability of its ssDNA substrates inside the cells and processes that produce ssDNA may stimulate transposition. We describe below a link between the transposition of these elements and the replication fork. Moreover, in the case of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''], single strand DNA produced during re-assembly of the ''[[wikipedia:Deinococcus_radiodurans|D. radiodurans]]'' genome following irradiation results in stimulation of transposition<ref><pubmed>20090938</pubmed></ref><ref name=":17"><pubmed>16359337</pubmed></ref>. Transcription or other processes leading to horizontal gene transfer such as transformation, conjugative transfer, or transduction with single strand [[wikipedia:Bacteriophage|phages]] might also favor their mobility. 

=====Replication fork=====
The replication fork modulates the transposition of many transposable elements ([https://tncentral.ncc.unesp.br/report/te/Tn7-NC_002525 Tn''7''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS903 IS''903''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS10R IS''10''], [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS50R IS''50''], [https://tncentral.ncc.unesp.br/report/te/Tn4430-X07651.1 Tn''4430''], [[wikipedia:P_element|P element]]<ref><pubmed>19703395</pubmed></ref><ref><pubmed>9620951</pubmed></ref><ref><pubmed>3000598</pubmed></ref><ref><pubmed>2451025</pubmed></ref><ref><pubmed>2546858</pubmed></ref><ref><pubmed>21896744</pubmed></ref>. For IS''200''/IS''605'' family members, the replication fork, in particular the lagging strand template, is an important source of ss DNA substrates for both excision and integration. Transposition can be considered to follow a “Peel and Paste ” mechanism ([[:File:Fig. IS200.16.png|Fig. IS200.16]]) where the IS excises or is “peeled” off as a single strand circle from the lagging strand template of the donor molecule and then integrates or is “pasted” in a ss target at the replication fork.
[[Image:Fig. IS200.16.png|thumb|center|720x720px|'''Fig. IS200.16.''' '''Top''': Excision of the single-strand circular intermediate (transposon joint) from the lagging strand template of a donor plasmid. Arrow tip: replication direction.  '''Bottom''': Integration of right end (RE)–left end (LE) transposon joint into the single-strand target at the replication fork.|alt=]]

'''Excision:''' Excision of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] is sensitive to the direction of replication across the element: it is more frequent when the active strand (top strand) is on the lagging strand (discontinuous) template ([[:File:Fig. IS200.16.png|Fig. IS200.16]] '''top'''; [[:File:Fig. IS200.17.png|Fig. IS200.17]]) but difficult to detect when it is on the leading (continuous) strand <ref name=":25" />. Moreover, excision in vitro requires that both ends are in single strand form at the same time <ref name=":24" />.
[[Image:Fig. IS200.17.png|thumb|center|600x600px|'''Fig. IS200.17.''' Orientation with respect to replication direction. The disposition of the IS608 active (top) strand with respect to replication direction is shown when the fork approaches from one direction (left) when it is part of the lagging-strand template or the other (right) when it is part of the leading strand. [[wikipedia:Okazaki_fragments|Okazaki fragments]] on the lagging strand are indicated as short lines. The direction of DNA synthesis is indicated with half arrowheads.|alt=]]

The length of ssDNA on the lagging-strand template depends on the initiation frequency of [[wikipedia:Okazaki_fragments|Okazaki fragment]] synthesis by the [[wikipedia:DnaG|DnaG primase]]<ref><pubmed>1531480</pubmed></ref><ref><pubmed>1740453</pubmed></ref>. Transient inactivation of [[wikipedia:DnaG|DnaG]] activity reduces this frequency and therefore increases the average length of ssDNA between [[wikipedia:Okazaki_fragments|Okazaki fragments]]; the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] excision frequency increased. Under permissive conditions for ''[[wikipedia:Escherichia_coli|E. coli]]'' carrying a dnaGts mutation, using a plasmid-based assay with [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] derivatives of different lengths, the excision frequency decreased strongly as IS length increased. In contrast, when DnaGts activity was reduced by growth under sub-lethal conditions, excision showed a much less pronounced length-dependence ([[:File:Fig. IS200.18.png|Fig. IS200.18]]). This length-dependence might also contribute to the difference in copy numbers observed in the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] groups (see "[[IS Families/IS200-IS605 family#Distribution and Organization|Distribution and Organization]]").
[[Image:Fig. IS200.18.png|thumb|center|600x600px|'''Fig. IS200.18.''' '''IS''608'' Excision of as a function of IS length (in kilobases). Bottom panel:''' Shows the effect of IS length on the frequency of excision '''using IS608 derivatives of''' 0.3; 0.5; 0.8; 1.1; 1.4; 1.9; 3 and 4 kb. Excision frequency falls steeply with increasing length and assumes a lower length dependence for IS of greater than about 2kb. In a dnaGts strain at the permissive temperature of 33°C, excision is significantly reduced as a function of length (X). Taken from ton-Hoang et al. 2010.|alt=]]

'''Integration:''' [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] integration is oriented (with its left end 3’ to a TTAC target site) and it requires an ssDNA target ''in vitro <ref name=":13" />''<ref name=":7" />. The close link between transposition and the replication fork is also illustrated by the integration bias, consistent with a preference for an ssDNA target on the lagging strand template ([[:File:Fig. IS200.16.png|Fig. IS200.16]] '''bottom'''). This was indeed found to be the case in ''E. coli'' for both plasmid and chromosome targets <ref name=":25" />. As expected, the orientation of insertions into the ''E. coli'' chromosome was correlated with the direction of replication of each replicore and was consistent with integration into the lagging strand template. 

The orientation bias is not restricted to [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']. An ''in silico'' analysis of a large number of bacterial genomes carrying copies of various family members revealed that most had a strong insertional bias consistent with the direction of replication <ref name=":25" /> ([[:File:Fig. IS200.19.png|Fig. IS200.19]]). Moreover, in certain cases, elements which did not follow the orientation pattern could be correlated to the genomic region that had undergone inversion or displacement ([[:File:Fig. IS200.20.png|Fig. IS200.20]]; [[:File:Fig. IS200.21.png|Fig. IS200.21]]) suggesting that, once they occur, insertions are quite stable. It seems possible that this type of genomic archaeology based on orientation patterns could be used to complement the study of bacterial genome evolution.[[Image:Fig. IS200.19.png|thumb|center|600x600px|'''Fig. IS200.19.''' The orientation of IS200/IS605 family members in different bacterial genomes.

Overall GC skew (G – C / G + C) is indicated in blue and orange. Top.  ''[[wikipedia:Salmonella_enterica|S. enterica]]'' (typhi) CT18; 

'''Middle.'''  ''[[wikipedia:Yersinia_pseudotuberculosis|Y. pseudotuberculosis]]'' IP31758; '''Bottom'''. ''[[wikipedia:Photobacterium_profundum|P. profundum]]'' SS9. Replication is bidirectional from a single origin (red spot). Arrows top or bottom indicate the point and orientation of insertion.|alt=]][[Image:Fig. IS200.20.png|thumb|center|720x720px|'''Fig. IS200.20.''' Orientation of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1541 IS''1541''] in ''[[wikipedia:Yersinia_pestis|Yersinia pestis]]'' (Microtus).

Overall GC skew (G – C / G + C) is indicated in blue and orange. Replication is bidirectional from a single origin (red spot). Arrows top or bottom indicate the point and orientation of insertion. The IS orientation adheres strictly to the GC skew, suggesting that there have been many chromosome rearrangements after IS insertion. |alt=]]
[[Image:Fig. IS200.21.png|thumb|center|720x720px|'''Fig. IS200.21.''' '''Comparison of ''S. enterica'' (typhi) CT18 and Ty2 genomes.''' The two ''[[wikipedia:Salmonella_enterica|S. enterica]]'' genomes are known to contain a large inversion generated by recombination between two rRNA operons (Deng et al., 2003). This is illustrated by the circular map at the bottom of the figure (from Deng et al., 2003). The top of the figure shows the positions of inversion with respect to the origin of replication. The two replicores are shown in blue and orange. The multiple copies of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] are shown as black vertical arrowheads. Those pointing upwards indicate [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] in one orientation, while those pointing downwards indicate the opposite orientation. |alt=]]

'''Stalled replication forks:''' Stalled replication forks appeared preferential targets for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] insertion. In the experiments using the Tus/ter replication termination or operator/repressor system, replication fork arrest attracts [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] insertion <ref name=":25" />. Transient blockade of the unidirectional replication fork by the Tus protein at the ter site resulted in preferential [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] insertion into the array of target sequences behind the stalled forks on the lagging strand but not on the leading strand ([[:File:Fig. IS200.22.png|Fig. IS200.22]]). A similar result was obtained in the ''[[wikipedia:Escherichia_coli|E. coli]]'' chromosome using the ''lacI''/''lacO'' and ''tetR''/''tetO'' repressor/operator roadblock systems<ref><pubmed>12864855</pubmed></ref><ref name=":34"><pubmed>27466393</pubmed></ref> ([[:File:Fig. IS200.23.png|Fig. IS200.23]]). Moreover, a significant number of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] insertions into the ''[[wikipedia:Escherichia_coli|E. coli]]'' chromosome were localized in the highly transcribed rrn operons. This suggests that high transcription levels might affect replication fork progression (fork arrest by collision with RNA polymerase, R-loop formation, etc.) and could account for targeting the rrn operons. Thus, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] insertions can be targeted to the stalled forks and this may well represent a major pathway for targeting transposition.
[[Image:Fig. IS200.22.png|thumb|center|700x700px|'''Fig. IS200.22.''' Map of insertions with ter in the permissive and non-permissive orientations. Replication from d’ori is from left to right; * = target sequences TTAC close to ter ; horizontal arrow heads = Ternp (red) et Terp (black) ; vertical black arrow heads = IS''608'' insertions, vertical red arrow heads = multiple [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] insertions upstream of Ternp and within Terp. (Ton-Hoang et al., 2010).|alt=]]
[[Image:Fig. IS200.23.png|thumb|center|700x700px|'''Fig. IS200.23.''' '''Top''': Position of the ''lacO'' and ''tetO'' arrays in ''E.coli'' WX45 and WX51: The replication origin, ori, is shown as a red ellipse and the left and right replicores in blue and orange respectively. ''E.coli'' WX45 and WX51 contain arrays at different locations. Bottom: Insertions into ''E.coli'' WX45 and WX51; the left and right replicores have been separated for convenience. '''Above''': a detail of the ''lacO'' array (light orange or light green rectangles) on the left replicore. '''Below''': a detail of the ''tetO'' array (orange or green rectangles) on the right replicore. Black vertical arrows: insertions obtained in the absence of LacI (top) or TetR (bottom). Green or orange vertical arrows: insertions obtained in the presence of LacI (top) or TetR (bottom) in several independent experiments. The positions of the oligonucleotides (not to scale) used to localize the insertions are shown with half arrowheads. The [[wikipedia:Kanamycin_A|kanamycin]] and [[wikipedia:Gentamicin|gentamycin]] resistance cassettes used in the construction and insertion of the lac and tet operator arrays are also shown. * represents potential TTAC target sequences present in the region.|alt=]]

=====Genome re-assembly after irradiation in ''[[wikipedia:Deinococcus_radiodurans|D. radiodurans]]''=====
''[[wikipedia:Deinococcus_radiodurans|Deinococcus radiodurans]]'', arguably the most radiation-resistant organism known, has a remarkable capacity to survive the lethal effects of DNA-damaging agents, such as ionizing radiation, UV light and desiccation. After exposure to high irradiation doses, the ''[[wikipedia:Deinococcus_radiodurans|D. radiodurans]]'' chromosome which is present in multiple copies per cell<ref><pubmed>649572</pubmed></ref><ref><pubmed>7309705</pubmed></ref> is shattered and degraded, but can be very rapidly reassembled in a process called ESDSA ('''E'''xtended '''S'''ynthesis '''D'''ependent '''S'''trand '''A'''nnealing). This involves resection of the multiple dsDNA fragments to generate extensive ssDNA segments, reannealing of complementary DNA and reconstitution of the intact chromosome<ref name=":26" />.

Mennecier et al.<ref name=":17" /> analyzed the mutational profile in the ''thyA'' gene following irradiation. The majority of mutants were due to the insertion of a single IS, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] which is present in a single copy in the genome of the laboratory ''D. radiodurans'' strain. Furthermore, using a tailored genetic system, both [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] excision and insertion efficiency was found to increase significantly following host cell irradiation<ref><pubmed>20090938</pubmed></ref>. A PCR-based approach was used to follow irradiation-induced excision of the single genomic [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] copy and re-closure of flanking sequences. Remarkably, these events are temporally closely correlated with the start of the ESDSA. The signal that triggers IS''Dra2'' transposition is likely the production of ssDNA intermediates generated during genome reassembly. Consistent with this, the requirement of ssDNA substrates for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''], as for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''], was confirmed by ''in vitro'' studies of TnpAIS''Dra2''-catalysed cleavage and strand transfer<ref><pubmed>20090938</pubmed></ref>.

[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] excision also depends on the direction of replication and is consistent with a requirement for the active strand to be located on the lagging strand template in normally growing cells. However, this bias disappeared in irradiated ''[[wikipedia:Deinococcus_radiodurans|D. radiodurans]] <ref name=":25" />''. Since no apparent strand bias was observed in generating ssDNA during ESDSA, the lack of orientation bias in irradiated [[wikipedia:Deinococcus_radiodurans|''D. radiodurans'']] suggests that ssDNA substrates are no longer limited to those rendered accessible during replication. This indicates that ssDNA sources are different in the contexts of vegetative replication and in genome reassembly.

=====Real-time transposition (excision) activity=====
The dynamics of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] excision from a donor site has been examined at the colony and single-cell level in real-time using an artificial [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] derivative inserted between the -35 and -10 elements of a PlacIQ1 promoter<ref><pubmed>27298350</pubmed></ref> driving expression of the blue fluorescent protein mCerulean<ref><pubmed>21479270</pubmed></ref>. TnpAIS''608'', N-terminally tagged with the bright yellow reporter Venus<ref><pubmed>11753368</pubmed></ref> was supplied in trans driven by PLTetO1 and controllable over a 100x range. Excision rates were proportional to the transposase levels and, as expected, excision depended on the orientation of the IS derivative with respect to the direction of replication in the donor plasmid: IS in an orientation with the active IS strand in the lagging strand template excised more frequently and at lower (10x) TnpA levels than when inserted into the leading strand, demonstrating the validity of the experimental system. In this system, individual excision events as bright flashes of blue fluorescence. Following an initial activity in the part of the population when cells are applied to a solid medium, activity decreases or ceases during “exponential” growth but increases again at a constant rate (in a sub-population) upon growth arrest in a random ([[wikipedia:Poisson_distribution|Poisson distributed]]) way. Moreover, the events do not occur randomly in the growing colonies and tend to be excluded from the colony edges. The study underlines the heterogeneity of TE activity rates in both space and time possibly resulting from heterogenous TnpA levels at the individual cell level in the population. These studies are reminiscent of the early studies of [[wikipedia:James_A._Shapiro|Jim Shapiro]] on [[wikipedia:Bacteriophage_Mu|phage Mu]]-mediated rearrangements in growing bacterial colonies<ref><pubmed>2838063</pubmed></ref><ref><pubmed>2553666</pubmed></ref>.

===TnpB===
TnpA alone can carry out both the cleavage and joining steps ''in vitro''. TnpB is encoded only by the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] groups and is not required for transposition of either [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] or [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] in ''[[wikipedia:Escherichia_coli|Escherichia coli]]'' and ''[[wikipedia:Deinococcus_radiodurans|Deinococcus radiodurans]]'' respectively<ref name=":13" /><ref name=":24" /><ref name=":38"><pubmed>20090938</pubmed></ref>. The full length TnpB is approximately 400 amino acids long. An overview of TnpB organization was originally obtained by comparing the entire [https://isfinder.biotoul.fr/ ISfinder] collection of 85 ''tnpB'' copies with the [https://pfam.xfam.org/ Pfam domain database] ([[:File:Fig. IS200.24.png|Fig. IS200.24]]).  
<br />[[Image:Fig. IS200.24.png|thumb|center|540x540px|'''Fig. IS200.24.''' Organization of TnpB protein and derivatives: putative N-terminal [[wikipedia:Helix-turn-helix|helix-turn-helix motif (HTH)]], central OrfB_IS''605'' domain with a putative DDE motif (Pfam), and C-terminal [[wikipedia:Zinc_finger|zinc finger motif (ZF)]] are shown. Numbers represent the occurrence of corresponding variants among 85 analyzed sequences: 46 carry all the three domains (e.g., [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']), 33 lack the [[wikipedia:Helix-turn-helix|HTH motif]] (e.g., [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608'']), whereas others retain separate domains.|alt=]]

====IS''200''/IS''605'' and the ISC group====
As described above ([[:File:Fig. IS200.1.png|Fig. IS200.1]]) IS''200''/IS''605'' family members fall into 4 categories: those which carry both ''tnpA'' and ''tnpB'' genes either in a divergent or in a collinear configuration ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605'']-like); those which lack ''tnpB'' and carry only ''tnpA'' ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200'']-like); and those which lack ''tnpA'' altogether and include only ''tnpB'' ([https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like; <ref name=":22" /><ref name=":34" />). Members of the latter class are presumably unable to undergo autonomous transposition.

Among the approximately 400 entries in [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder] (November 2021), there are more than 200 examples of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like derivatives and about 50 examples [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200'']-like derivatives. It was suggested that the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like derivatives might undergo transposition using a resident ''tnpA'' gene to supply a Y1 transposase ''in trans'', a suggestion which remains to be formally demonstrated <ref name=":34" />. There is some circumstantial evidence for transposition of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like elements. For example, [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS891 IS''891''], present in multiple copies in the cyanobacterium ''[[wikipedia:Anabaena|Anabaena]]'' sp. strain M-131 genome was observed to have inserted into a plasmid which had been introduced into the strain <ref name=":33" />.

The lack of an essential role for TnpB is underlined by the finding that other members of the IS''200''/IS''605'' family carry ''tnpB'' genes which have undergone (or are undergoing) decay <ref name=":18"><pubmed>23461641</pubmed></ref>.

====ISC, the IS''200''/IS''605'' related IS which carry IscB, a Cas9-related alternative to TnpB.====
Another group of potential IS of similar organisation, the ISC insertion sequence group, was defined by Kapitonov et al.<ref name=":29"><pubmed>PMC4810608</pubmed><br /></ref> following identification of [[wikipedia:Cas9|Cas9]] homologues which occur outside the [[wikipedia:CRISPR|CRISPR]] structure, so called “stand-alone” homologues. While related to TnpB, they are more similar to [[wikipedia:Cas9|Cas9]] than to TnpB proteins. These genes were often flanked by short DNA sequences which, like '''LE''' and '''RE''' of the IS''200''/IS''605'' family, were capable of forming secondary structures. Moreover, it was reported that the ends of many ISC derivatives showed significant identity to members of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] derivatives identified by these authors in the same study. ([[:File:FigIS200.25.png|Fig. IS200.25]]). These structures therefore resemble the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like group. Note that the secondary structures and their function in TnpA-mediated transposition have been demonstrated only for [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISHp608 IS''Dra2'']. 
[[File:FigIS200.25.png|alt=|center|thumb|600x600px|'''Fig. Tn200.25.''' Potential secondary structures in IS''200''/IS''605''/ISC ends. For the IS''200/''IS''605'' members, the sequences of the left ('''LE''') and right ('''RE''') ends are shown in red and blue respectively.  Note that these structures have only been verified for I[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS608 IS''608''] and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISHp608 IS''Dra2''] The other sequences are from Kapitonov et al. <ref name=":29" />. The potential secondary structures are indicated by horizontal blue arrows and bold type face.]]


These potential transposable elements were called '''ISC''' (Insertion Sequences Encoding [[wikipedia:Cas9|Cas9]]; not to be confused with [https://tncentral.ncc.unesp.br/TnPedia/index.php/IS_Families/IS91-ISCR_families#ISCR IS''CR'', IS with '''C'''ommon '''R'''egion]). The name IscB was coined for the [[wikipedia:Cas9|Cas9-like]] protein and IscA for an associated potential transposase protein which was identified in a very limited number of cases. Examples of ISC elements with both ''iscA'' and ''iscB'' genes are quite rare. Only 7 cases were identified by Kapitonov et al.,<ref name=":29" /> ([[:File:FigIS200.26.png|Fig. IS200.26]]) and only 56 of 2811 ''iscB'' examples observed in a more extensive analysis were accompanied by an ''iscA'' copy <ref name=":30"><pubmed>34591643</pubmed></ref> . Most ISC identified were [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like with only the ''iscB'' (''tnpB''-like) gene. These stand-alone IscB copies were identified in multiple copies in a large number of bacterial and archaeal genomes generally in low numbers (<10 copies) although some genomes contained more elevated numbers (e.g. 22 in ''[[wikipedia:Methanosarcina|Methanosarcina lacustris]]''; 25 in ''[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=64178 Coleofasciculus chthonoplastes]'' PCC 7420; 52 in ''[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=363277&lvl=3&lin=f&keep=1&srchmode=1&unlock Ktedonobacter racemifer]'')<ref name=":29" />.

However, in contrast to the observations of Kapitonov et al.,<ref name=":29" /> more wide-ranging studies <ref name=":30" /> identified rare IscB proteins which were not “stand alone” but were associated with [[wikipedia:CRISPR|CRISPR]] arrays (31 examples in a sample of 2811).

A tree of “full-length” elements ([[:File:FigIS200.26.png|Fig. IS200.26]]; <ref name=":29" />)(i.e. those with both ''tnpA'' and ''tnpB'' or ''iscB'' genes) based on TnpA/IscA sequences showed that full length [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] and ISC examples carrying both ''tnpA''/''iscA'' and ''tnpB''/''iscB'' are interleaved. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] is among those family members with divergent ''tnpA'' and ''tnpB'' genes ([[:File:Fig. IS200.1.png|Fig. IS200.1]]) while other family members carry ''tnpA'' upstream of ''tnpB'' (e.g. [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']). However, in contrast to all [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605'']-like derivatives, those full length ISC elements included in this tree all have the ''iscA'' gene downstream of and slightly overlapping with ''iscB''.[[File:FigIS200.26.png|alt=|center|thumb|600x600px|'''Fig. IS200.26.''' Phylogenetic tree of Y1 transposases encoded by IS''605'' (TnpA) and IS''C2Y'' (IscA). From Kapitonov et al. <ref name=":29" />. TnpA: RED; IscA: LIGHT RED; TnpB: GREY; IscB: BLUE. The arrowheads indicate the direction of expression. The IS were identified in: '''KR''', ''[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=363277 Ktedonobacter racemifer]'' DSM44963; '''CS''', ''[[wikipedia:Coprobacillus|Coprobacillus]]'' sp. 3_3_56FAA; '''EC''', ''[[wikipedia:Enterococcus|Enterococcus cecorum]]'' DSM 20682 (ATCC 43198); '''AA''', ''[[wikipedia:Anaeromusa_acidaminophila|Anaeromusa acidaminophila]]'' DSM 3853; '''CH''', ''[[wikipedia:Clostridium_novyi|Clostridium. haemolyticum]]'' NCTC 9693; '''MA''', ''[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=313606 Microscilla marina]'' ATCC 23134; '''VB''', ''[https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=553239 Vibrio breoganii]''; '''BC''', ''[[wikipedia:Bacteroides|Bacteroides coprophilus]]''; '''MM''', ''[[wikipedia:Methanosarcina|Methanosarcina mazei]]''; '''MZ''', ''[[wikipedia:Methanosalsum|Methanosalsum zhilinae]]''; '''EH''', ''[[wikipedia:Anaerobutyricum_hallii|Eubacterium hallii]]'' DSM 3353; '''BSp''', ''[[wikipedia:Butyrivibrio|Butyrivibrio]]'' sp MB2005; '''BMT2''', ''[[wikipedia:Bacillus|Bacillus]]'' sp. MT2; '''FP''', ''Francisella philomiragia''; '''HP''', ''[[wikipedia:Helicobacter_pylori|Helicobacter pylori]]'' Hp H-16; '''RI''', ''[[wikipedia:Roseburia_inulinivorans|Roseburia inulinivorans]]''.]]

====ISC have very similar transposases to those of the IS''200''/IS''605'' family and are therefore part of the same super family.====
An alignment of full length TnpA from the IS''200''/IS''605'' group ([[:File:FigIS200.26.png|Fig. 200.26]]; [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder] November 2021) shows the highly conserved '''HuH''' triad, catalytic tyrosine ('''Y''') and important glutamine ('''Q''') residues all central to the transposition chemistry ([[:File:Fig. IS200.6.png|Fig. IS200.6]], [[:File:Fig. IS200.11.png|Fig. IS200.11]] and [[:File:Fig. IS200.12.png|Fig. IS200.12]]) together with a number of other highly conserved amino acid positions. An alignment with the available IscA from the ISC group ([[:File:FigIS200.27.png|Fig. 200.27]]  '''Top''') shows that these also include all the highly conserved TnpA amino acid positions and are therefore very closely related to TnpA. However, the IscA and TnpA proteins appear to fall into separate clades ([[:File:FigIS200.27.png|Fig. 200. 27]] '''bottom''') with some overlap.
[[File:FigIS200.27.png|alt=|center|thumb|600x600px|'''Fig. IS200.27.''' Alignment of TnpA proteins from the IS''200''/IS''605'' family. The data is drawn from ISfinder (November 2021). The alignment was performed with Clustal omega2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/) and drawn using Jalview Version 2. The '''HuH''', '''Y''' and '''Q''' residues are indicated. A consensus sequence is included beneath.]]
Since IS families are defined by their transposases rather than their accessory genes, and those of ISC and the IS''200''/IS''605'' family are so similar, it seems reasonable to include the ISC group as a subgroup of the IS''200''/IS''605'' family (or [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS605 IS''605''] super family;<ref name=":29" />). For many of the archaeal elements, there is a small, potential 40-45 amino acid, peptide located upstream of the TnpB analogue.

<br />
[[File:FigIS200.28.png|alt=|center|thumb|640x640px|'''Fig. IS200.28.''' Alignment of TnpA proteins from the IS''200''/IS''605'' family with IscA. The sequences for IS''200''/IS''605'' family members are from [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder] (November 2021) and the IscA proteins from Kapitonov et al.<ref name=":29" /> kindly supplied by [[wikipedia:Kira_Makarova|Kira Makarova]]. '''Top.''' The alignment was performed with Clustal omega2 and drawn using Jalview Version 2. The '''HuH''', '''Y''' and '''Q''' residues are indicated. A consensus sequence is included beneath. The IscA proteins from Kapitonov et al. are included. '''Bottom.''' Phylogenetic tree from the same alignment. The Sequences from Kapitonov et al. <ref name=":29" />are boxed.]]
A tree based on the TnpB/IscB ([[:File:FigIS200.28.png|Fig. IS200.28]]) examples presented by Kapitonov, et al.,.<ref name=":29" /> shows that the TnpB homologues form a clade separate from IscB and that the latter can be divided into two clades, IscB1 and IscB2.

These considerations therefore reinforce the idea that the IS''200''/IS''605'' family and ISC group might be considered as a superfamily which includes a number of related accessory genes (''tnpB'', ''iscB1'', ''iscB2'' etc), which carry flanking DNA sequences with secondary structure potential and in which a Y1 HuH transposase assures the chemistry of transposition. A similar conclusion was also reached by Altae-Tran et al.<ref name=":30" /> .However, this picture is complicated by the identification of another group of transposable elements, the [https://tncentral.ncc.unesp.br/TnPedia/index.php/IS_Families/IS607_family IS''607'' family] in which ''tnpB'' is associated with a different type of transposase, in this case a serine site-specific recombinase ([https://tncentral.ncc.unesp.br/TnPedia/index.php/IS_Families/IS607_family IS''607'' family]).

<br />
[[File:FigIS200.29.png|alt=|center|thumb|720x720px|'''Fig. IS200.29.''' Tree of IscB and TnpB proteins from Kapitonov et al.<ref name=":29" />. Boxed segments show major clades. Note that the ''iscB'' proteins fall into 3 principal clades which are separated from ''tnpB''.]]
<br />

====TnpB, IscB, Cas12 and Cas9====
Clearly, TnpB and IscB form distinct groups ([[:File:FigIS200.29.png|Fig. IS200.29]]). Full length TnpB is approximately 400 amino acids long. An overview of TnpB organization was initially obtained by comparing an early [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder] collection of 85 ''tnpB'' copies with the [http://pfam.xfam.org/ Pfam domain database] ([[:File:Fig. IS200.24.png|Fig. IS200.24]]). This revealed three major domains: an N-terminal putative [[wikipedia:Helix-turn-helix|helix-turn-helix]], a longer and more variable central domain, OrfB_IS605, with a putative '''DDE motif''' and a C-terminal [[wikipedia:Zinc_finger|zinc finger (ZF) domain]] of the '''CPXCG''' type. Half of the analyzed TnpB copies including TnpBIS''Dra2'' but not TnpBIS''608'' contained all three domains, while only two did not include a [[wikipedia:Zinc_finger|zinc finger]].

Pasternak et al.'''<ref name=":18" />''' observed that TnpB appears to have an inhibitory effect on [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] excision and insertion in its host, ''[[wikipedia:Deinococcus_radiodurans|D. radiodurans]]'', and on excision in ''[[wikipedia:Escherichia_coli|E. coli]]'', and that the integrity of its putative [[wikipedia:Zinc_finger|zinc finger motif]] is required for this effect. Interestingly, analysis of the [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder database] revealed that IS''200''/IS''605'' family elements exhibiting a high genomic copy number often encode TnpA alone.

TnpB has been identified in both prokaryotes and eukaryotes. It is carried by members of the [https://tncentral.ncc.unesp.br/TnPedia/index.php/IS_Families/IS607_family IS''607'' family] found both in prokaryotes and in eukaryotes and their viruses but is dispensable for [https://tncentral.ncc.unesp.br/TnPedia/index.php/IS_Families/IS607_family IS''607'' transposition] in ''[[wikipedia:Escherichia_coli|E. coli]]'' . As it is for IS''200''/IS''605'' transposition. TnpB analogues, known as '''Fanzor1''' and '''Fanzor2''', have also been identified in diverse eukaryotic transposable elements. TnpB/Fanzor proteins may function as transposition regulatory proteins ''in vivo''.

====TnpB and IscB are Related to the RNA-guided nucleases [[wikipedia:CRISPR/Cas12a|Cas12]] and [[wikipedia:Cas9|Cas9]].====
More extensive analysis showed that TnpB shares some similarity with the RNA-guided nuclease [[wikipedia:CRISPR/Cas12a|Cas12]] while IscB shows greater similarity to [[wikipedia:Cas9|Cas9]]. Both, like [[wikipedia:Cas9|Cas9]] and [[wikipedia:CRISPR/Cas12a|Cas12]], exhibit split [[wikipedia:RuvABC|RuvC endonuclease domains]] <ref><pubmed>24728998</pubmed></ref> <ref name=":29" /><ref><pubmed>PMC5851899</pubmed></ref><ref><pubmed>31857715</pubmed></ref><ref name=":37"><pubmed>34619744</pubmed></ref> ([[:File:FigIS200.30.png|Fig. IS200.30]]). While [[wikipedia:Cas9|Cas9]] and [[wikipedia:CRISPR/Cas12a|Cas12]] carry related functional domains, their architectures are somewhat different and the configuration of their guide RNAs also differ.

<br />
[[File:FigIS200.30.png|alt=|center|thumb|680x680px|'''Fig. IS200.30.''' Schematic of IscB and TnpB showing the relative positions of the different functional motifs. '''Top:''' IscB (Extracted from Altae-Tran et al.<ref name=":30" />). Botton: TnpB from [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] compared with the Cas12 derivative, Un1Cas12f1 (from Karvelis, et al.<ref name=":37" />). [Green]: [[wikipedia:RuvABC|RuvC]] segment '''I''', '''II''' and '''III'''; [red]: [[wikipedia:Zinc_finger|Zinc Finger]] or HNH nuclease; [blue]: Arginine rich helix; [yellow]: Wedge domain; [grey]: Helical bundle. As defined by  Altae-Tran et al.<ref name=":30" /> (IscB) and TnpB Karvelis, et al.<ref name=":37" />. Note, compared to Cas9-like IscB, TnpB and Cas12 have an N-terminal extension before the first [[wikipedia:RuvABC|RuvC]] ('''i''') motif. ]]

====IscB and Cas9====
[[wikipedia:Cas9|Cas9]] (also called Cas5, Csn1, or Csx12) is an RNA-guided dual nuclease generally associated with [[wikipedia:CRISPR|CRISPR systems]] in bacteria and widely used in genome engineering. The [[wikipedia:RuvABC|RuvC DED catalytic]] triad is split into three sections (I, II and III) in which I and II are interrupted by the R-rich region and II and III by an [https://proteopedia.org/wiki/index.php/H-N-H_motif HNH nuclease] domain. A region common to all [[wikipedia:Cas9|Cas9]] derivatives is located at the C-terminal end.

The [[wikipedia:Cas9|Cas9]] structure has been determined ([[:File:FigIS200.31.png|Fig. IS200.31]]. '''B''' <ref name=":39"><pubmed>24505130</pubmed></ref>). The protein is a monomer in which the three RuvC segments I, II and II carrying the D, E and D catalytic residues respectively, are assembled into the correct three-dimensional configuration to generate a [[wikipedia:RuvABC|RuvC-like]] catalytic pocket with the [https://proteopedia.org/wiki/index.php/H-N-H_motif HNH nuclease] domain extruded ([[:File:FigIS200.31.png|Fig. IS200.31]]. '''A'''). The [[wikipedia:Cas9|Cas9]] guide RNA (crRNA) is composed of a region containing secondary structure potential and a 5’ extension (spacer) of about 20 nts, complementary to the target sequence and which forms an RNA/DNA [[wikipedia:Heteroduplex|heteroduplex]] ([[:File:FigIS200.31.png|Fig. IS200.31]]. '''C'''). Activated [[wikipedia:Cas9|Cas9]] recognises a specific sequence, PAM ('''P'''rotospacer '''A'''djacent '''M'''otif), located next to the target sequence on the complementary strand downstream of the target sequence. This is necessary for binding of the [[wikipedia:Cas9|Cas9]]-crRNA complex and subsequent cleavage <ref name=":36"><pubmed>22949671</pubmed></ref>. Cleavage is catalysed by both the HNH nuclease (target strand) and the reconstituted [[wikipedia:RuvABC|RuvC nuclease]] (complementary strand). Cleavage is often “blunt” (i.e. occurs at the same position on both strands) and PAM proximal <ref name=":36" />.

<br />
[[File:FigIS200.31.png|alt=|center|thumb|680x680px|'''Fig. IS200.31.''' '''[[wikipedia:Cas9|Cas9]] Structure and Activity.''' '''A)''' Cartoon of [[wikipedia:Cas9|Cas9]] showing the “assembled” RuvC domains. (colors as in legend to Fig. Tn200. YD. '''B)''' [[wikipedia:Cas9|Cas9]] structure from Jinek et al. <ref name=":39" /> . Taken from https://en.wikipedia.org/wiki/Cas9#/media/File:Cas9_Apo_Structure.png. The [[wikipedia:RuvABC|RuvC]] and HNH endonuclease domains are indicated. '''C)''' Mechanism of [[wikipedia:Cas9|Cas9]] action. The target DNA is invaded by 3' end of the guide RNA and cleavage of the PAM-carrying strand is accomplished by the [[wikipedia:RuvABC|RuvC]] segments of Cas12 while cleavage of the RNA-bound opposite strand is assisted by the [[wikipedia:Zinc_finger|zinc-finger domain]]. ]]


IscB shares [[wikipedia:Cas9|Cas9]] sequence features such as the split [[wikipedia:RuvABC|RuvC]] and [https://proteopedia.org/wiki/index.php/H-N-H_motif HNH nuclease] domains and an arginine-rich (R-rich) domain ([[:File:FigIS200.30.png|Fig. IS200.30]] '''Top''') with a group of [[wikipedia:Cas9|Cas9]] derivatives, Cyan7822_6324, in particular <ref><pubmed>21756346</pubmed></ref>. In addition, a more detailed investigation  <ref name=":30" /> led to identification of an additional IscB N-terminal domain (called PLMP after its conserved amino acid residues) not present in [[wikipedia:Cas9|Cas9]] ([[:File:FigIS200.30.png|Fig. IS200.30]]. '''Top'''). These features can be clearly observed in alignments of IscB sequences of Kapitonov <ref name=":29" /> ([[:File:FigIS200.32.png|Fig. IS200.32]]).

<br />
[[File:FigIS200.32.png|alt=|center|thumb|720x720px|'''Fig. IS200.32.''' Alignment of IscB. Sequences from Kapitonov et al. <ref name=":29" />. The alignment was performed with Clustal omega2 and drawn using Jalview Version 2. PLMP (Altae-Tran et al. <ref name=":30" />), RuvC I, II and II, ariginine rich region (R-rich) and HN(H) motifs are indicated as well as a CXXC zinc finger. A consensus sequence is included beneath.]]

====TnpB and [[wikipedia:CRISPR/Cas12a|Cas12]]====
[[wikipedia:CRISPR/Cas12a|Cas12]] is also an RNA-guided nuclease. Twelve subtypes have been described <ref><pubmed>31021231</pubmed></ref> and the structures of a number of these have been solved. They have similar C-terminal ends but carry (related) N-terminal ends of various lengths (see Karvelis, et al.<ref name=":37" />). One of the shorter derivatives [[wikipedia:CRISPR/Cas12a|Cas12F]] (AKA Cas14) <ref><pubmed>33764415</pubmed></ref> acts as a dimer. Like [[wikipedia:Cas9|Cas9]], the common C-terminal end is composed of a split [[wikipedia:RuvABC|RuvC]] (I, II and III) in which I and II are interrupted by the R/K-rich region. In this case, however, instead of the HNH domain, [[wikipedia:RuvABC|RuvC]] segments II and III are separated by a [[wikipedia:Zinc_finger|zinc finger]] of the CPXCG typeI ([[:File:FigIS200.32.png|Fig. IS200.32]] '''bottom'''). 

For [[wikipedia:CRISPR/Cas12a|Cas12]], the guide RNA is composed of a region containing secondary structure potential and a 3’ extension (spacer) of about 20 nts, complementary to the target sequence ([[:File:FigIS200.33.png|Fig. IS200.33]]). The PAM sequence is located upstream of the target sequence. Cleavage is PAM distal and staggered.
[[File:FigIS200.33.png|alt=|center|thumb|640x640px|'''Fig. IS200.33.''' '''Cas12 Structure and Activity. A)''' A comparison of the structure of Cas12f1 with the model of TnpB (kindly provided Karvelis et al. <ref name=":37" />) showing the REC and WED domains (left) and the helical, [[wikipedia:RuvABC|RuvC]] and [[wikipedia:Zinc_finger|Zn-finger domains]] (right). '''B) Mechanism of Cas12 action.''' The target DNA is invaded by the 3'end of the guide RNA and cleavage of the PAM-carrying strand is accomplished by the [[wikipedia:RuvABC|RuvC]] segments of Cas12 while cleavage of the RNA-bound opposite strand is assisted by the [[wikipedia:Zinc_finger|zinc finger domain]].  ]]


Karvelis, et al.<ref name=":37" /> describe the domain structure of TnpB and present evidence that it is related to [[wikipedia:CRISPR/Cas12a|Cas12]], another derivative of the Cas family ([[:File:FigIS200.30.png|Fig. IS200.30]] '''bottom'''). Like [[wikipedia:CRISPR/Cas12a|Cas12F]], it also carries a [[wikipedia:RuvABC|RuvC]] in which the '''D''' (I), '''E''' (II) and '''D''' (III) catalytic residues are split. Again, [[wikipedia:RuvABC|RuvCI]] and [[wikipedia:RuvABC|RuvCII]] are separated by an R-rich region and [[wikipedia:RuvABC|RuvCII]] and [[wikipedia:RuvABC|RuvCII]] by a [[wikipedia:Zinc_finger|zinc finger]] with three modules ([[:File:Fig. IS200.24.png|Fig. IS200.24]]; [[:File:FigIS200.30.png|Fig. IS200.30]]). Moreover, the N-terminal region which corresponds to the minimal common structural elements present in [[wikipedia:CRISPR/Cas12a|Cas12]]  <ref name=":37" />, includes a three helical bundle Rec domain (labelled [[wikipedia:Helix-turn-helix|HTH]] in an earlier TnpB analysis; [[:File:Fig. IS200.24.png|Fig. IS200.24]]), inserted into a [[wikipedia:Beta_barrel|β-barrel domain]], referred to as the “Wedge” domain in [[wikipedia:CRISPR/Cas12a|Cas12]].

These features can be identified in an alignment of the entire TnpB library (349 examples from [https://tncentral.ncc.unesp.br/ISfinder/index.php ISfinder]; November 2021) ([[:File:FigIS200.34i.png|Fig. IS200.34]] '''i''', '''ii''' and '''iii''') and in TnpB sequences provided by Kapitonov et a.,<ref name=":29" /> ([[:File:FigIS200.35.png|Fig. IS200.35]]). <gallery mode="slideshow">
File:FigIS200.34i.png|'''Fig. IS200.34.''' TnpB Alignment
File:FigIS200.34ii.png|'''Fig. IS200.34.''' TnpB Alignment
File:FigIS200.34iii.png|'''Fig. IS200.34.''' TnpB Alignment
</gallery>

The relationship between [[wikipedia:CRISPR/Cas12a|Cas12]] and TnpB has strong support from structural modelling <ref name=":37" />: for example Un1Cas12f1 (Cas14a) from an uncultured archeon <ref><pubmed>33333018</pubmed></ref>, which functions as an asymmetric dimer and represents a minimal domain organization of the [[wikipedia:CRISPR/Cas12a|Cas12]] group <ref name=":37" />. However, TnpB from [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] (see below) appears to be a monomer <ref name=":37" />. [[File:FigIS200.35.png|alt=|center|thumb|650x650px|'''Fig. IS200.35.''' TnpB Alignment with RuvC and Other Domains. Sequences from Kapitonov et al <ref name=":29" />. The alignment was performed with Clustal omega2 and drawn using Jalview Version 2 The long N-terminal extension, RuvC I, II and II, arginine/lysine rich region (RK-rich) and [[wikipedia:Zinc_finger|zinc finger motifs]] are indicated <ref name=":37" />.]]
<br />'''Evolution of TnpB and IscB from an Ancestral RuvC.'''

In view of the relationship between TnpB, IscB, [[wikipedia:RuvABC|RuvC]] and the Cas proteins, the important question of the evolutionary trajectory of these proteins arises. Using various analytic tools, it was concluded that all [[wikipedia:Cas9|Cas9]] examples identified to date are probably descended from a single IscB derivative ancestor <ref name=":30" />. This contention arose from the observation that the CRISPR-associated IscB derivatives do not form a single clade but are distributed over the IscB phylogenetic tree suggesting that they evolved independently from a single acquisition <ref name=":30" />. Additional IscB derivatives were also identified in this study which led to an evolutionary scenario involving successive acquisition of domains by an ancestral [[wikipedia:RuvABC|RuvC]] ([[:File:FigIS200.36.png|Fig. IS200.36]]). The additional species included a shorter derivative, IsrB, which carried the bridging helix but not the [https://proteopedia.org/wiki/index.php/H-N-H_motif HNH domain] and a longer derivative which had acquired a so-called REC domain <ref name=":30" />.

TnpB appears to have followed an alternative evolutionary route towards [[wikipedia:CRISPR/Cas12a|Cas12]]. In addition, it is thought that TnpB was an ancestor of the eukaryotic Fanzor proteins <ref name=":35"><pubmed>23548000</pubmed></ref> associated with diverse eukaryotic potential transposable elements.

<br />
[[File:FigIS200.36.png|alt=|center|thumb|640x640px|'''Fig. IS200.36.''' S'''equential Acquisition of Domains by an Ancentral RuvC from Altae-Tran et al.'''<ref name=":30" />. [green]: [[wikipedia:RuvABC|RuvC]] segments '''I''', '''II''' and '''III'''; [red]: [[wikipedia:Zinc_finger|Zinc-finger]] or HNH nuclease; [blue]: Arginine rich helix; [yellow]: Wedge domain. As defined by Altae-Tran et al.<ref name=":30" /> (IscB) and TnpB Karvelis et al. <ref name=":37" />. Note, compared to Cas9-like ''iscB'', TnpB and Cas12 have an N-terminal extension before the first [[wikipedia:RuvABC|RuvC]] (i) motif while iscB carries the N-terminal P domain [grey].  ]]
====Functional analysis of TnpB and IscB====
Clearly, the relationship between TnpB and IscB and [[wikipedia:CRISPR/Cas12a|Cas12]] and [[wikipedia:Cas9|Cas9]] respectively suggested that TnpB and IscB might function as RNA guided nucleases which may, in some way, be involved in transposition <ref name=":37" /><ref name=":30" /> and this has been extensively tested.

====TnpB functions as an RNA-guided Endonuclease.====
For TnpB, Karvelis, et al.<ref name=":37" /> used [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] as a model system. This has the advantage that its transposition behavior has been well characterised <ref name=":38" />'''<ref name=":18" />'''.

In [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''], the 3’ end of the upstream ''tnpA'' gene overlaps the 5’ end of ''tnpB''. The authors were unable to efficiently express TnpB as a fusion protein but observed that its yield was significantly increased when in its natural context but in which TnpA had been inactivated by mutation. Although the nature of the mutation is not specified in the article, its behavior could be explained if it were an in-frame deletion or other mutation which does not affect C-terminal translation. Since, ([[:File:FigIS200.37.png|Fig. IS200.37]]) it seems likely that expression of TnpB involves translational coupling  <ref><pubmed>7517937</pubmed></ref><ref><pubmed>PMC6728339</pubmed><br /></ref> with TnpA.
[[File:FigIS200.37.png|alt=|center|thumb|600x600px|'''Fig. IS200.37.''' Organization of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']. '''Top:''' Map of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] showing the left ('''LE''') and right ends ('''RE''') (red and blue respectively), the 5' pentanucleotide target sequence, '''TTGAT''', the position of cleavage indicated by the vertical arrowheads and the overlapping ''tnpA'' and ''tnpB'' genes. Above is shown the DNA and protein sequences at the position of the overlap, which are presumably involved in translational coupling of ''tnpA'' and ''tnpB''. '''Botton:''' Sequence of the guide RNA (reRNA) derived from the right IS end including a few bases of the IS interior, the '''RE''' secondary structure (blue) and the IS flank which acts the guide (green) from Karvelis et al. <ref name=":37" />. ]]
TnpB was found to purify with RNA of approximately 150 nts derived from the '''IS''' RE (reRNA). reRNA was complementary to the ''tnpB'' 3’ end, RE and about 16 nt of (host) flanking DNA ([[:File:FigIS200.38.png|Fig. IS200.38]]). This RNA with the secondary structure provided by the RE sequence and the 3’ extended flanking DNA is of the expected configuration for relatives of [[wikipedia:CRISPR/Cas12a|Cas12]] ([[:File:FigIS200.36.png|Fig. IS200.36]]). Previous studies had identified non coding RNA (ncRNA) from the 3’ end of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''], a related IS from ''[[wikipedia:Halobacterium_salinarum|Halobacterium salinarum]]'' NRC-1, called sense overlapping transcripts (sotRNAs) <ref name=":31"><pubmed>PMC4615843</pubmed></ref>.
[[File:FigIS200.38.png|alt=|center|thumb|600x600px|'''Fig. IS200.38.''' Defining TAM and the Position of Cleavage. '''A) Experimental design'''. A plasmid encoding TnpB [purple] and an reRNA with a 16 (or 20) defined flank sequence [green] terminated by a specific  [[wikipedia:Hepatitis_delta_virus_ribozyme|Hepatitis delta virus ribozyme]], HDV [black], (left) was used to produce the TnpB-reRNA complex and a lystate was used to treat a plasmid library containing the defined flank sequence with an upstream heptanucleotide of random sequence [red]. Double-strand cleavage products were captured by adapter ligation. '''B) Library DNA sequence.''' Randomised nucleotides are shown in red, guide sequence in green. '''C) Preferred TAM sequence and cleavage.''' The preferred TAM sequence, the observed pentanucleotide '''[https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2'']''' target sequence TTGAT, is shown in red and the cleavages observed are shown by vertical arrowheads <ref name=":37" />.]]


Such sotRNA transcripts, specific for ''tnpB'' genes, had previously been identified by Gomes-Filho et al., <ref name=":31" /> in a number of Archaea and Bacteria including ''[[wikipedia:Sulfolobus_acidocaldarius|S. acidocaldarius]]'', ''[[wikipedia:Methanopyrus|Methanopyrus kandleri]]'', ''[[wikipedia:Helicobacter_pylori|Helicobacter pylori]]'' and ''[[wikipedia:Escherichia_coli|E. coli]]'' K12. There has also been some indication of “transposase-related” sense overlapping transcripts of ''tnpB''-like genes from [[wikipedia:Thermococcus_kodakarensis|''T. kodakarensis'']] <ref><pubmed>PMC4247193</pubmed></ref> and ''[[wikipedia:Pyrococcus_furiosus|P. furiosus]]'', <ref><pubmed>PMC124278</pubmed></ref>. However, that these may represent guide RNAs had not been explicitly considered.

Karvelis et al.<ref name=":37" /> demonstrated that TnpB, purified using a '''His''' tag, could cleave DNA. They argued that since the 3’ end of the [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] reRNA corresponds to the DNA target, it would vary according to the position of the IS insertion and the reRNA may (have) serve(d) as a guide RNA. If true, cleavage of the target DNA should occur within the 3’ extension sequence of the flank (the foot of '''RE''' in [[:File:FigIS200.37.png|Fig. IS200.37]]). In this context, it is interesting that the (DNA) structure of the right end was shown to form a base triple which is a characteristic of RNA <ref name=":7" />. 

To determine whether RNA-guided cleavage occurred , they constructed a system ([[:File:FigIS200.38.png|Fig. IS200.38]]. '''A''') using a plasmid supplying TnpB together with an reRNA which included a 16 (or 20) defined flank sequence and was terminated by a specific [[wikipedia:Hepatitis_delta_virus_ribozyme|Hepatitis delta virus ribozyme]] (HDV; <ref><pubmed>9288893</pubmed></ref>) to produce a defined 3’ RNA end <ref><pubmed>9783582</pubmed></ref>. A lysate from the host strain was then used in cleavage assays of a library of target plasmids each containing a specific defined 16 base pair sequence directly downstream from a 7 bp (7N) randomised sequence ([[:File:FigIS200.38.png|Fig. IS200.38]]. '''B'''). This has previously been used to identify conserved PAM sequences  <ref name=":37" /> <ref><pubmed>30691644</pubmed></ref>. Specific double strand cleavage products were captured by adapter ligation (details in Karvelis et al.<ref name=":37" />  and the sequence of the resulting enriched 7N region was determined. This corresponded to the conserved [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] target pentanucleotide '''TTGAT''' (with a higher enrichment for GA) sequence which is essential for IS insertion and abuts '''LE''' in the integrated IS. By equivalence to PAM, this sequence was called TAM ('''T'''ransposon '''A'''djacent '''M'''otif) <ref name=":37" /> see also <ref name=":30" />.

This cleavage specificity was confirmed using purified TnpB-RNP in which the protein and RNA components were produced by separate plasmids and a target plasmid carrying a 3’ flank and a 5’ TTGAT TAM pentanucleotide and a different guide sequence ([[:File:FigIS200.38.png|Fig. IS200.38]].'''C'''). The results showed a majority of double strand breaks in the supercoiled target plasmid to generate linear plasmids but also a significant level of nicked product. The TnpB-RNP was also active on a linear substrate. In both cases, use of a TnpB D191A mutant, part of the conserved [[wikipedia:RuvABC|RuvC]] DED catalytic triad, eliminated the reaction. Robust TnpB-mediated cleavage activity was observed and required both TAM and guide RNA sequences. Further sequence analysis revealed that cleavage occurred distal to the TAM sequence at the guide sequence boundary and was specific for cleavage on the bottom strand but showed some variation on the top strand ([[:File:FigIS200.38.png|Fig. IS200.38]]). There are some differences however with Cas12. TnpB is a monomer and requires a single copy of reRNA <ref name=":37" />.
[[File:FigIS200.39.png|alt=|center|thumb|600x600px|'''Fig. IS200.39.''' Cas9 cleavage was: TAM (PAM) proximal (3 nts from TAM for the target and 8 and 12 nts for the non-target strands; that the RNP protected DNA from [[wikipedia:Exonuclease_III|ExoIII digestion]] 19 nts upstream of the TAM on the target and 6 downstream on the non-target strand. ]]


Moreover, ''in vivo'', TnpB expression together with reRNA from one plasmid resulted in loss of a second plasmid carrying the reDNA target (interference), presumably as a result of cleavage at the target site and linearisation of the plasmid. This of course may explain the inhibitory effect of TnpB originally observed by Pasternak et al. '''<ref name=":18" />'''.

Additionally, the authors were able to demonstrate that the system functions in eukaryotic cells opening the possibility that it could be suitably modified for gene editing.

A similar study by Altae-Tran et al.<ref name=":30" /> using purified TnpB from a less well characterised ''tnpB'' gene of ''[[wikipedia:Alicyclobacillus_macrosporangiidus|Alicyclobacillus macrosporangiidus]]'', showed that the protein catalysed cleavage of both double- and single-stranded DNA targets in both a TAM-dependent and TAM independent manner. As in the case of TnpB<sub>IS''Dra2''</sub>, ''[[wikipedia:Alicyclobacillus_macrosporangiidus|A. macrosporangiidus]]'' TnpB-associated guide RNA was identified and derived from the 3’ end of the ''tnpB'' gene. In this case, the TAM appeared to be the tetranucleotide TCAC.

====IscB functions as an RNA-guided Endonuclease====
Altae-Tran et al.<ref name=":30" /> also examined a very large number of rather disperse IscB systems for their endonuclease properties, their association with RNA and their capacity as RNA guide proteins. Initial studies concerned a [[wikipedia:CRISPR|CRISPR]] associated IscB (marked in the article as Delaware Bay acquatic sample), which when purified from a heterologous ''[[wikipedia:Escherichia_coli|Escherichia coli]]'' host was associated with an RNA localised directly upstream of ''iscB'' which generated a signal in a PAM “discovery” assay and was able to generate cleavage products ''in vitro'' with the appropriate target. 

An alignment of over 500 (non-redundant) ''iscB'' genes revealed an upstream region of conserved sequence of about 300 bp which terminated at what the authors state is an IS''200''/IS''605''-like end. One specific example examined, present in the host ''[http://microbes.sites.haverford.edu/LaboratoryWiki/Ktedonobacter_racemifer K. racemifer]'' genome in nearly 50 copies, was associated with non-coding RNA species in most cases, which they called ΩRNA, with significant secondary structure potential. An example of ''[http://microbes.sites.haverford.edu/LaboratoryWiki/Ktedonobacter_racemifer K. racemifer]'' IscB was investigated ''in vitro'' using a plasmid substrate and shown to: use a target adjacent pentanucleotide TAM, ATAAA; and that cleavage was reprogrammable by changing the complementary RNA extension (guide).

To further characterise IscB, the TAM sequences of 57 examples from a collection of 86 genes from a phylogenetically diverse set of bacteria could be determined; of those 57, 5 were reconstituted with their wRNA and found to active in target cleavage; and one, AwaIscB from ''[[wikipedia:Allochromatium|Allochromatium warmingii]]'', was chosen for further study.  

Biochemically, AwaIscB could cleave double strand DNA in a magnesium dependent reprogrammable way with a temperature optimum of 35-40°C and with RNA guide lengths of between 15 and 45 nts. A mutation of the [[wikipedia:RuvABC|RuvC]] E residue eliminated cleavage of the non-target strand while mutation of an '''H''' residues in the '''HNH''' motif eliminated cleavage of the target strand (as expected for a Cas9-related enzyme; [[:File:FigIS200.31.png|Fig. IS200.31]]). Mutation of both residues eliminated cleavage altogether. Also like Cas9, cleavage was: TAM (PAM) proximal (3 nts from TAM for the target and 8 or 12 nts for the non-target strands; that the RNP protected DNA from ExoIII digestion 19 nts upstream of the TAM on the target and 6 downstream on the non-target ([[:File:FigIS200.39.png|Fig. IS200.39]]); and that truncation of the newly identified N-terminal PLMP domain ([[:File:FigIS200.40.png|Fig. Tn200.40]] '''top''') eliminated activity. 
[[File:FigIS200.40.png|alt=|center|thumb|680x680px|'''Fig. IS200.40.''' '''TnpB-Facilated Replacement of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] at the Donor Joint by Copy-Choice. A)''' Model Proposed by Karvelis et al. <ref name=":37" />. From left to right: The replication fork generates single-strand DNA on the lagging strand which reveals a single strand circular IS copy allowing the intervention of the TnpA transposase. This liberates a single strand circular IS copy and creates a donor joint in which the '''LE''' target sequence and the '''RE''' flanking DNA are joined. TnpB together with the guide RNA produced from the intact IS copy targets the donor joint and introduces a double-strand cleavage (red arrowheads). The broken strands then invade the intact IS and generate a new IS copy by copy choice.  '''B) Possible copy-choice mechanism.''' Invasion of the sister chromatid carrying the intact IS by the 3' end created from the donor joint double-strand break, providing primers for replicative copying. LE and RE are shown in red and blue respectively, the RE flank in green. Newly replicated DNA is shown as dotted line.  ]]

====A Model for TnpB Function During Transposition====
The role of these RNA-guided endonucleases remains unknown. It has been suggested that they may mediate RNA-guided TnpA-catalysed transposition although it has been shown that TnpA together with the transposon ends contain all the information for integration into single stranded DNA (references in Mobile DNA paper) and TnpB is not required for transposition. Another suggestion is that they may represent a toxin-type system <ref name=":30" />.

While there is considerable information concerning the IS''200''/IS''605'' family, very little is known about the mobility of structures which include IscB. Although there is circumstantial evidence that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341''] related elements are mobile, there is no formal evidence that IscB-containing structures are mobile. The rarity of full length ISC has hampered functional studies.

However, one of the important remaining questions concerning Tn''200''/Tn''605'' transposition pathway was the way in which these IS increase their copy number following peeling off the donor daughter “chromatid” to leave a transposon-less donor joint. In the model shown in [[:File:Fig. IS200.16.png|Fig. IS200.16]]., excision of a single stranded circular IS intermediate from the lagging strand leaves one double strand copy of the IS in the donor replicon carried by one of the daughter “chromatids” (on the leading strand) and a “donor joint” on the other. In this scenario, no increase in the IS copy number would occur.

The results of Karvelis et al.<ref name=":37" /> with [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] provide a solution for this problem. The authors propose a model in which, following excision of the single strand circular IS copy and formation of the donor joint ([[:File:FigIS200.40.png|Fig. IS200.40]] '''A'''), reRNA/TnpB targeted cleavage of the donor joint is used to initiate a copy/choice replacement of the IS from the remaining IS on the daughter chromatid ([[:File:FigIS200.40.png|Fig. IS200.40]] '''A'''). This would permit maintenance of donor replicon integrity while assuring an increase in IS copy number via transposition. They point out that conceptually, this in some ways resembles group I intron behavior <ref><pubmed>24589358</pubmed></ref>.

TnpA-mediated transposition of the IS''200''/IS''605'' family is well documented. The transposition model it is based on in vitro experiments using single-strand oligonucleotides, results from ''in vivo'' experiments which implicate [[wikipedia:DnaG|DnaG]] and the observation that the orientation of insertion is correlated with the direction of replication of the target chromosome (see <ref><pubmed>26350330</pubmed></ref> and references therein). The proposed function of TnpB in this process neatly completes the transposition model by offering an explanation of how IS copy number is maintained by replacement of the excised IS at the resulting donor joint. However, it is puzzling that [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS1341 IS''1341'']-like elements of both the TnpB and IscB type have proliferated extensively compared to the “full length” IS. This raises the question of the way in which these genetic objects arose and their function in the cell. 

Future studies will be necessary to determine whether they are able to transpose and/or recombine with or without a TnpA-like transposase.

<br />

===Y1 transposase domestication===
There are many examples of eukaryotic transposases whose activities have been appropriated to perform various cellular functions (see <ref><pubmed>16937363</pubmed></ref><ref><pubmed>23935529</pubmed></ref><ref><pubmed>24348275</pubmed></ref>. However, the very few examples of this domestication for prokaryotic enzymes concern Y1 transposases.

====TnpAREP and REP/BIME====
Recently, a new clade of Y1 transposases (TnpAREP) was found associated with REP/BIME sequences in structures called '''REPtrons''' <ref name=":19"><pubmed>20085626</pubmed></ref><ref name=":27"><pubmed>22199259</pubmed>
</ref> ([[:File:Fig. IS200.7.png|Fig. IS200.7]] '''A'''). In spite of their compact size, bacterial genomes carry many repetitive sequences, often important for genome function and evolution. Among them, '''R'''epetitive '''E'''xtragenic '''P'''alindromic sequences (or REPs) are short DNA repeats of 20-40 bp that can form stem-loop structures preceded by a conserved tetranucleotide (GTAG or GGAG) ([[:File:Fig. IS200.25.png|Fig. IS200.41]]). REPs are found in intergenic regions in many bacterial species, particularly in proteobacteria, at high copy number <ref name=":19" /><ref><pubmed>20528935</pubmed></ref><ref name=":20"><pubmed>23758774</pubmed>
</ref>. There are nearly 590 copies in ''[[wikipedia:Escherichia_coli|Escherichia coli]]'' K12<ref name=":40"><pubmed>2092362</pubmed></ref> ([[:File:FigIS200.42.png|Fig. IS200.42]]) and up to 2200 copies in ''[[wikipedia:Pseudomonas|Pseudomonas]]'' sp GM79<ref name=":20" />. REPs can exist as individual units but can cluster in more complex structures called '''B'''acterial '''I'''nterspersed '''M'''osaic '''E'''lements (BIME). These are composed of two individual REPs in inverse orientation (REP and iREP) separated by a short linker of variable length. BIME are often found in consecutive tandem copies ([[:File:Fig. IS200.25.png|Fig. IS200.41]]). Several roles have been attributed to these sequences including genome structuring, post-transcriptional regulation and genome plasticity. REPs are known to interact with protein partners such as [[wikipedia:Bacterial_DNA_binding_protein#IHF|Integration Host Factor]]<ref><pubmed>8262044</pubmed></ref>, [[wikipedia:DNA_gyrase|DNA gyrase]]<ref><pubmed>9427406</pubmed></ref> and [[wikipedia:DNA_polymerase#Pol_I|DNA polymerase I]]<ref><pubmed>2197600</pubmed></ref>. REPs also increase mRNA stability and can act as transcriptional terminators<ref><pubmed>20528935</pubmed></ref><ref><pubmed>14731278</pubmed></ref> or as targets for different IS <ref name=":22" /><ref><pubmed>16563168</pubmed></ref>. It has also been suggested that REP sequences are involved in REP sequences can downregulate translation of upstream genes dependent on trans-translation. This occurs only if they are within 15 nt of a termination codon. It has been suggested that that REPs can stall ribosomes, leading to mRNA cleavage and induction of the trans-translation process<ref><pubmed>25891074</pubmed></ref>. Recombination at REP sequences has also been shown to be involved in the formation of F’ plasmid derivatives (the classic F plasmid carrying various portions of the chromosome ([[:File:FigIS200.43.png|Fig. IS200.43]]) from Hfr strains<ref><pubmed>12511513</pubmed></ref>. However, the origin of REPs and their dissemination mechanisms are poorly understood.
[[Image:Fig. IS200.25.png|thumb|center|720x720px|'''Fig. IS200.41.''' '''Top''': Representation of two categories of REP structures in ''E. coli''/''Shigella'' with mismatches in the hairpin stem in orange and light blue, violet box represents the conserved tetranucleotide GTAG. Corresponding iREP structures in red and dark blue where green box represents the complementary tetranucleotide CTAC. '''(ii)''' Structure of BIME: REP and iREP separated by linkers C or D. BIME are frequently found as consecutive copies. '''Bottom''': '''(iii)''' Examples of REPtrons from some representative ''E. coli'' strains. tnpAREP is shown in gray, the flanking genes ''yafL'' and ''fhiA'' in green and in violet, respectively. Arrows represent the direction of transcription.|alt=]]
[[File:FigIS200.42.png|alt=|center|thumb|720x720px|'''Fig. IS200.42. Rep/BIME distribution round the ''E. coli'' K12 chromosome.''' Data is from Bachellier 1999 <ref name=":40" />. The number of symbols at a given map position indicates the number of tandem copies of each element. Details on the sequence of the different BIMEs can be found in Bachellier 1999 <ref name=":40" />.]]
[[File:FigIS200.43.png|alt=|center|thumb|720x720px|'''Fig. IS200.43. Rep sequences involved in the formation of F’ primes.''' Adapted from Slechta and Roth 2003. Integration of plasmid F and its excision to generate F’128. Filled arrows, genes; open arrows, insertion sequences. Gray arrows designate nested or recombinant insertion sequences. The arc inside the map at the right indicates the extent of pOX38, a minimal F plasmid derivative still capable of both vegetative replication and conjugation. Insertion occurs by homologous recombination between an IS''3'' copy located on the chromosome and the resident IS''3'' copy on plasmid F. Excision occurs between two Rep sequences located downstream of the ''mhp'' locus and upstream of the ''dinB'' locus on the ''E. coli'' chromosome.]]
Although more complex, REPtrons are reminiscent of [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=IS200 IS''200''] group members ([[:File:Fig. IS200.25.png|Fig. IS200.41]]). However REPtrons do not appear to be mobile and, in general, a single copy of a given REPtron co-exists with numerous corresponding REP/BIME and genomes may harbor several distinct REPtrons<ref name=":19" /><ref name=":20" />. It has therefore been suggested that REP/BIMEs represent a special type of non-autonomous transposable element mobilizable by TnpAREP.

In vitro analysis of REPtrons: Analysis of ''[[wikipedia:Escherichia_coli|E. coli]]'' REPtron activity in vitro has shown that, like TnpAIS''200''/IS''605'', TnpAREP strictly requires single stranded REP/BIME DNA substrates and is strand specific, only REP can be processed, whereas iREP are refractory to cleavage <ref name=":27" />. Purified ''E. coli'' TnpAREP promotes ssREP cleavage (in the linker sequences either 3’ or 5’ to the REP structure) and rejoining, and this activity requires the conserved tetranucleotide GTAG and the bulge in the middle of the REP stem <ref name=":27" /><ref name=":21"><pubmed>22885300</pubmed>
</ref>. Cleavage in vitro is less specific than that of TnpAIS''200''/IS''605'' and occurs at a CT dinucleotide. 

In contrast to TnpAIS''608'' and TnpAIS''Dra2'', ''[[wikipedia:Escherichia_coli|E. coli]]'' TnpAREP is a monomer in solution and in the crystal structure<ref name=":21" />. Moreover, in the co-crystal structure, the short C-terminal tail is inserted into the active site blocking access to an ssDNA. It may, therefore, play a regulatory role in the activity. Indeed C-terminal truncation of TnpAREP resulted in increased cleavage activity relative to the full-length protein ''in vitro''. The biochemical and structural analysis suggested that the GTAG 5’ to the foot of the REP hairpin may play a similar role to the guide sequences GL/R in IS''200''/IS''605''. Moreover, structural data also highlighted numerous specific contacts between TnpAREP and GTAG, explaining its importance in the activity and clearly distinguishing TnpAREP from TnpAIS''200''/IS''605'', which do not directly contact the guide sequences (Cleavage site recognition). The way by which TnpAREP promotes REP/BIME proliferation through their host genomes remains to be determined. 

====IStrons====
Another role for Y1 transposases was suggested by the identification of chimeric genetic elements widely distributed in the genome of ''[[wikipedia:Clostridium_difficile|Clostridium difficile]]''<ref name=":6"><pubmed>10931294</pubmed></ref> and ''[[wikipedia:Bacillus_cereus|Bacillus cereus]]'' group<ref><pubmed>16907808</pubmed></ref><ref><pubmed>18587153</pubmed></ref><ref name=":28"><pubmed>16030238</pubmed></ref>: IStrons. These combine functional and structural properties of [[wikipedia:Group_I_catalytic_intron|group I introns]] at their 5’-end with those of an IS element at their 3’-end ([[:File:FigIS200.44.png|Fig. IS200.44]]). This 3' part contains an IS''200''/IS''605'' related sequence including two full length or truncated orfs, ''tnpA'' and ''tnpB'', very similar to those found in [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISDra2 IS''Dra2''] (''[[wikipedia:Deinococcus_radiodurans|D. radiodurans]]'') and [https://tncentral.ncc.unesp.br/ISfinder/scripts/ficheIS.php?name=ISCpe2 IS''Cpe2''] (''[[wikipedia:Clostridium_perfringens|C. perfringens]]''). IStrons are present at several loci in the same genome, indicating that this element is mobile and may move as a complete genetic unit. All IStron copies analyzed so far are inserted 3’ to the pentanucleotide '''TTGAT'''. ''In vivo'', all variants can be efficiently and precisely excised signifying that components necessary for ribozyme activity are present <ref name=":6" />. Little is known about IStron behavior but the data suggest that IS components could mediate the spread of IStron while the intron component could assure splicing. 
[[File:FigIS200.44.png|alt=|center|thumb|600x600px|'''Fig. IS200.44.''' Organization of IStron where Intron and IS parts are indicated. P1–P8 and IGS represent characteristic features of [[wikipedia:Group_I_catalytic_intron|group I Introns]]. '''LE''', '''RE''', '''TTGAT''' target site and two orf of the IS part are indicated.]]
''In vitro'' oligonucleotide-based assays using purified IStron transposase confirmed that at the DNA level, TTGAT is the LE cleavage site in excision and the target site respectively ([https://www-lmgm.biotoul.fr/uk/equipes/grpieva/equipe.html Caumont-Sarcos], unpublished). At the RNA level, the same sequence is probably required in the splicing reaction<ref><pubmed>19667762</pubmed></ref>. This would represent a novel type of intron invasion and transposition mechanism and provide a direct link between RNA and DNA worlds.

It is interesting to note that related IStrons have recently been identified which include components of the [[IS Families/IS607 family|IS''607'' family]] <ref name=":28" /><ref><pubmed>25324310</pubmed></ref>. These are characterised by a serine transposase together with a ''tnpB'' gene <ref name=":14" />.

==Bibliography==
<references />