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• W2023259875 abstract "DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the “top” IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication. DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the “top” IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication. IS200/IS605 family single-stranded DNA transposition is coupled to replication forks Replication direction through the IS plays a crucial role in activating IS excision Helicase or primase inhibition stimulates IS excision from the lagging strand Insertion shows a lagging-strand preference in normal and blocked forks DNA transposition involves movement of discrete DNA segments (transposons) from one genomic location to another. It occurs in all kingdoms of life and has contributed significantly to evolution of eukaryotes and prokaryotes. Transposable elements can represent a significant proportion of their host genomes (Biémont and Vieira, 2006Biémont C. Vieira C. Genetics: junk DNA as an evolutionary force.Nature. 2006; 443: 521-524Crossref PubMed Scopus (439) Google Scholar). They have been particularly well studied in bacteria where they are major motors of broad genome remodeling, play an important role in horizontal gene transfer, and can sequester and transmit a variety of genes involved in accessory cell functions, such as resistance to antimicrobial agents, catabolism of unusual compounds, and pathogenicity, virulence, or symbiosis. They are also important as genetic tools in identifying specific gene regulatory regions by insertion and are being developed as delivery systems for gene therapy applications. A variety of structurally and mechanistically distinct enzymes (transposases) have evolved to carry out transposition by several different pathways (Turlan and Chandler, 2000Turlan C. Chandler M. Playing second fiddle: second-strand processing and liberation of transposable elements from donor DNA.Trends Microbiol. 2000; 8: 268-274Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, Curcio and Derbyshire, 2003Curcio M.J. Derbyshire K.M. The outs and ins of transposition: from Mu to Kangaroo.Nat. Rev. Mol. Cell Biol. 2003; 4: 865-877Crossref PubMed Scopus (206) Google Scholar). They all possess an endonuclease activity allowing them to cleave, excise, and insert transposon DNA into a new location. Depending on the system (Curcio and Derbyshire, 2003Curcio M.J. Derbyshire K.M. The outs and ins of transposition: from Mu to Kangaroo.Nat. Rev. Mol. Cell Biol. 2003; 4: 865-877Crossref PubMed Scopus (206) Google Scholar), different types of nucleophile can be used by transposases to attack a phosphorus atom of a backbone phosphodiester bond and cleave DNA. These include water (generally activated by enzyme-bound metal ions), a hydroxyl group at the 5′ or 3′ end of a DNA strand, or a hydroxyl group of an amino acid of the transposase itself, such as serine or tyrosine. Many mobile DNA elements move using a “cut-and-paste” mechanism by excision of a double-stranded copy from one genomic location and insertion at another. Recently, a family of bacterial insertion sequences (ISs), the IS200/IS605 family, has been found that uses a completely different pathway and an unusual transposase with a catalytic tyrosine (a Y1 transposase). Studies of one member, IS608 (Figure 1A ), provided a detailed picture of their transposition (Ton-Hoang et al., 2005Ton-Hoang B. Guynet C. Ronning D.R. Cointin-Marty B. Dyda F. Chandler M. Transposition of ISHp608, member of an unusual family of bacterial insertion sequences.EMBO J. 2005; 24: 3325-3338Crossref PubMed Scopus (57) Google Scholar, Ronning et al., 2005Ronning D.R. Guynet C. Ton-Hoang B. Perez Z.N. Ghirlando R. Chandler M. Dyda F. Active site sharing and subterminal hairpin recognition in a new class of DNA transposases.Mol. Cell. 2005; 20: 143-154Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, Guynet et al., 2008Guynet C. Hickman A.B. Barabas O. Dyda F. Chandler M. Ton-Hoang B. In vitro reconstitution of a single-stranded transposition mechanism of IS608.Mol. Cell. 2008; 29: 302-312Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Barabas et al., 2008Barabas O. Ronning D.R. Guynet C. Hickman A.B. Ton-Hoang B. Chandler M. Dyda F. Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection.Cell. 2008; 132: 208-220Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In vitro, this requires single-stranded DNA (ssDNA) substrates and is strand specific: only the “top” strand is recognized by the element-encoded transposase, TnpA, and is cleaved and transferred, whereas the “bottom” strand does not transpose. Excision of the top strand as a transposon circle with joined left and right ends is accompanied by rejoining of the DNA flanks. The circle junction then undergoes TnpA-catalyzed integration into an ssDNA target in a sequence-specific reaction. Insertion involves transfer of both the 5′ and 3′ ends of the single-strand circle junction into the ssDNA target. The left (5′) IS608 end always inserts specifically just 3′ of the tetranucleotide, 5′-TTAC-3′ (Kersulyte et al., 2002Kersulyte D. Velapatiño B. Dailide G. Mukhopadhyay A.K. Ito Y. Cahuayme L. Parkinson A.J. Gilman R.H. Berg D.E. Transposable element ISHp608 of Helicobacter pylori: nonrandom geographic distribution, functional organization, and insertion specificity.J. Bacteriol. 2002; 184: 992-1002Crossref PubMed Scopus (67) Google Scholar), which is also essential for subsequent transposition (Ton-Hoang et al., 2005Ton-Hoang B. Guynet C. Ronning D.R. Cointin-Marty B. Dyda F. Chandler M. Transposition of ISHp608, member of an unusual family of bacterial insertion sequences.EMBO J. 2005; 24: 3325-3338Crossref PubMed Scopus (57) Google Scholar). The obligatorily single-stranded nature of IS200/IS605 transposition in vitro raises the possibility that it is limited in vivo by the availability of its ssDNA substrates. A number of cellular processes generate or occur using ssDNA, including DNA repair, natural transformation, conjugative plasmid transfer, single-stranded phage infection, and replication (where the DNA serving as the template for Okazaki fragment synthesis on the lagging strand of the replication fork is single stranded). Here, we investigate the link between IS608 transposition and the availability of ssDNA during replication in vivo. Our results demonstrate that transposition of the IS200/IS605 family is closely integrated into the host cell cycle and takes advantage of the presence of ssDNA on the lagging-strand template at the replication fork for dissemination. We also show that IS608 transposition is affected by perturbing the fork: transitory inactivation of crucial replication proteins increased excision from the lagging-strand template, and stalling of the fork resulted in insertions directed to the lagging strand of the blocked fork. Our results also suggest that insertion and excision of the related element, ISDra2, also depends on the lagging-strand template in its host, the radiation-resistant bacterium Deinococcus radiodurans, and that this dependency can be abolished with irradiation. We have extended our analysis to a number of related IS200/IS605 elements in a variety of sequenced bacterial genomes. The results of this in silico analysis are also consistent with a strong bias of insertion into the lagging-strand template in these organisms. Together, the results demonstrate the importance of the lagging-strand template for IS608 and ISDra2 activity and suggest that all IS200/IS605 family members have evolved a mode of transposition that exploits ssDNA at the replication fork. To investigate whether replication direction affects IS608 transposition, we used a plasmid assay in E. coli to monitor the excision step of transposition (Ton-Hoang et al., 2005Ton-Hoang B. Guynet C. Ronning D.R. Cointin-Marty B. Dyda F. Chandler M. Transposition of ISHp608, member of an unusual family of bacterial insertion sequences.EMBO J. 2005; 24: 3325-3338Crossref PubMed Scopus (57) Google Scholar). In this assay, the IS-carrying plasmids included an IS608 derivative in which the tnpA and tnpB genes (Figure 1A) were replaced by a chloramphenicol resistance (CmR) cassette (Figure 1C). In one case, the active (top) IS608 strand was located in the lagging-strand template (pBS102), and in the second, the replication origin was inverted (Figures 1B and 1C), placing the transposionally active top strand on the leading-strand template (pBS144). A second compatible plasmid supplied TnpA in trans under control of plac (Figure 1C). After overnight growth, IS excision was monitored by detection of reclosed donor backbone molecules from which the IS had been deleted. As shown in Figure 1C, when the IS608 active strand was located on the lagging-strand template, the donor backbone species could be clearly identified along with the parental plasmid and the plasmid used to supply transposase (lane 2). However, when the replication origin was inverted and the active IS strand was located on the leading strand, formation of the excised donor backbone species was only barely detectable (lane 3). This effect of replication direction on IS608 transposition was also observed in mating-out assays (Galas and Chandler, 1982Galas D.J. Chandler M. Structure and stability of Tn9-mediated cointegrates. Evidence for two pathways of transposition.J. Mol. Biol. 1982; 154: 245-272Crossref PubMed Scopus (58) Google Scholar) that measure overall transposition frequency. We monitored transposition was monitored by following movement of IS608 from a nonmobilizable donor plasmid into a conjugative plasmid. When the IS608 active strand was on the lagging-strand template (Figure 1D, line 2), the transposition frequency was 5.6 × 10−5, but when it was on the leading-strand template, the frequency dropped 27-fold to 2.1 × 10−6 (line 3). To ensure that this was not due to possible changes in transcription resulting from the inversion introduced during cloning to switch the orientation of the replication origin (where bla was inverted together with ori), we inserted transcriptional terminators (Simons et al., 1987Simons R.W. Houman F. Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions.Gene. 1987; 53: 85-96Crossref PubMed Scopus (1298) Google Scholar) on either one (lines 4–5) or both (lines 6–7) sides of the IS608 derivatives to insulate them from impinging transcription. In these cases, the observed effect of replication direction on transposition frequency remained unchanged. Since IS608 excision is sensitive to replication direction, we asked whether it was affected by perturbation of the replication fork. We used two temperature-sensitive replication mutants: dnaG308ts, encoding a mutant DNA primase, DnaG (Wechsler and Gross, 1971Wechsler J.A. Gross J.D. Escherichia coli mutants temperature-sensitive for DNA synthesis.Mol. Gen. Genet. 1971; 113: 273-284Crossref PubMed Scopus (257) Google Scholar), and dnaB8ts, encoding a mutant of the essential replication fork DNA helicase, DnaB (Carl, 1970Carl P.L. Escherichia coli mutants with temperature-sensitive synthesis of DNA.Mol. Gen. Genet. 1970; 109: 107-122Crossref PubMed Scopus (192) Google Scholar). Replication in these mutants occurs at 30°C but is interrupted after a shift to the restrictive temperature of 42°C. Inhibition of either DnaG or DnaB activity is expected to increase the amount of ssDNA at the fork, principally on the lagging-strand template (Louarn, 1974Louarn J.M. Size distribution and molecular polarity of nascent DNA in a temperature-sensitive dna G mutant of Escherichia coli.Mol. Gen. Genet. 1974; 133: 193-200Crossref PubMed Scopus (5) Google Scholar, Fouser and Bird, 1983Fouser L. Bird R.E. Accumulation of ColE1 early replicative intermediates catalyzed by extracts of Escherichia coli dnaG mutant strains.J. Bacteriol. 1983; 154: 1174-1183Crossref PubMed Google Scholar, Belle et al., 2007Belle J.J. Casey A. Courcelle C.T. Courcelle J. Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest.J. Bacteriol. 2007; 189: 5452-5462Crossref PubMed Scopus (25) Google Scholar) (see also the Discussion). To test the effect of inhibition of DnaG and DnaB activities, we used a genetic screen in which a β-lactamase gene is interrupted by an IS608 derivative (pAM1, Figure S2; Experimental Procedures). TnpA-catalyzed precise excision (using TnpA provided in trans) results in reconstitution of the β-lactamase gene (Figure 2A ) and the appearance of ampicillin resistant (ApR) colonies. As shown in Figures 2Bi, 2Bii, and 2Biii for the wild-type and dnaB and dnaG mutants, respectively, after overnight growth at 30°C (a permissive temperature) without TnpA induction, the frequency of ApR colonies was low for the wild-type and mutant hosts (column a). Induction of TnpA expression resulted in a nearly 3-fold increase in excision with little difference between wild-type, dnaBts, and dnaGts strains (column b). However, when the growth protocol was modified to include a 30 min temperature shift to 42°C (and further incubation of 3 hr at 30°C to allow replication to recover), excision increased about 10- and 7-fold in the dnaB and dnaG mutants, respectively, compared to the wild-type (column d). Omission of the 42°C step resulted in indistinguishable basal levels for wild-type and mutant hosts (column c). Thus, inactivation of DnaB helicase function or inhibition of initiation of Okazaki fragment synthesis with a dnaGts mutation stimulated the excision step of transposition, consistent with the notion that ssDNA at the replication fork is a substrate for IS excision. If excision occurs at the replication fork and requires ssDNA, it seemed possible that IS length might influence excision frequency since the probability that both IS ends are within the single-stranded region of the lagging-strand template should decrease with increasing IS length. We therefore examined the effect of IS length in the excision assay by using a set of IS608 derivatives with varying spacing between the left end (LE) and right end (RE) and found that excision decreased strongly as a function of increasing IS length and that these frequencies were strongly modified in a strain carrying a dnaGts mutation. Increasing the IS length from 0.3 to 2 kb, resulted in a 7- to 10-fold decrease in excision frequency with a log-linear relationship, consistent with the notion that excision occurs more efficiently when both ends are located within the short single lagging-strand region of about 1.5–2 kb upstream of the first complete Okazaki fragment at the replication fork (see Johnson and O'Donnell, 2005Johnson A. O'Donnell M. Cellular DNA replicases: components and dynamics at the replication fork.Annu. Rev. Biochem. 2005; 74: 283-315Crossref PubMed Scopus (438) Google Scholar, for review). As the IS length was further increased, excision decreased only slightly, at least up to a transposon length of 4 kb (Figure 2C) with a possible inflection between 1.5 and 2 kb. The length of ssDNA on the lagging-strand template depends on the initiation frequency of Okazaki fragment synthesis, in turn determined by DnaG. Progressive inactivation of DnaG activity by growth of the dnaGts mutant at increasing but sublethal temperatures should reduce this frequency and increase the mean length of ssDNA at the replication fork upstream of the first complete Okazaki fragment. We therefore analyzed excision of the IS608-derived transposons in wild-type and dnaGts mutants at different temperatures (Figure 2D). While profiles were indistinguishable for the wild-type strain at 30°C and 33°C, the dnaGts mutant exhibited a generally higher excision frequency at 30°C and showed a lower length dependent slope, revealing that the replication fork is affected by the mutation even at the normal permissive temperature. However, an inflection still appeared to occur. At 33°C, excision increased significantly, particularly for the longer transposons. To further examine this, we cloned the wild-type dnaG allele downstream of the tnpAIS608 gene in the TnpA-providing plasmid so that both were under control of the same promoter (Experimental Procedures). When this plasmid, pBS179, was introduced into the dnaGts strain, it clearly suppressed the dnaGts defect at 33°C (Figure 2E). Moreover, when introduced into the wild-type dnaG strain, the excision frequencies were even further reduced and the length-dependant slope was increased (Figure 2F). The circular E. coli chromosome replicates bidirectionally from the replication origin, oriC. If IS608 insertions target the lagging-strand template, they should occur in one orientation on one side of ori and in the opposite orientation on the other. To test this, we isolated IS608 insertions in the E. coli chromosome using a temperature-sensitive plasmid as the IS donor and supplying TnpA in trans (Figure 3A ). Insertions were localized by an arbitrary PCR procedure (Experimental Procedures) followed by DNA sequencing. We observed a dramatic skew in strand specificity of IS608 insertion relative to the origin of replication, oriC. Insertions either in the left or right replicores were in the orientation expected for transposition into the lagging-strand template (Figure 3B). It is interesting to note that while insertions were distributed around the chromosome, many appeared in the vicinity of the highly transcribed rRNA genes (Table S1). We also obtained insertions into the TnpA donor plasmid in the same experiment. In contrast to the chromosome, this plasmid replicates unidirectionally and the IS608 insertion pattern was completely (Figure 3C) different. All occurred into only one strand, the lagging-strand template, a result that has strong statistical support (Figure 3 legend). In all cases, both plasmid and chromosome insertions occurred 3′ to a TTAC target, as expected. As these are distributed equally on both strands of the chromosome and the TnpA donor plasmid (data not shown), the observed strand biases for insertion cannot be explained by a bias in target sequence distribution. Since our accumulating data suggested that IS608 targets ssDNA at the replication fork, we asked whether insertion could also be observed into forks stalled at a predefined location. For this, we used the E. coli Tus/Ter system in which a Ter site, when bound by the protein Tus, strongly reduces replication fork progression through Ter in the nonpermissive orientation (Ternp) (see Bierne et al., 1994Bierne H. Ehrlich S.D. Michel B. Flanking sequences affect replication arrest at the Escherichia coli terminator TerB in vivo.J. Bacteriol. 1994; 176: 4165-4167Crossref PubMed Google Scholar) but not in the opposite, permissive, orientation (Terp) (Neylon et al., 2005Neylon C. Kralicek A.V. Hill T.M. Dixon N.E. Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex.Microbiol. Mol. Biol. Rev. 2005; 69: 501-526Crossref PubMed Scopus (109) Google Scholar). The assay (Figure 4A and Figure S1) used a suicide conjugation system (Demarre et al., 2005Demarre G. Guérout A.M. Matsumoto-Mashimo C. Rowe-Magnus D.A. Marlière P. Mazel D. A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha) conjugative machineries and their cognate Escherichia coli host strains.Res. Microbiol. 2005; 156: 245-255Crossref PubMed Scopus (225) Google Scholar) in which the unidirectionally replicating target plasmid was carried by a recipient strain with an inactivated chromosomal tus gene. The target plasmid carried a functional tus gene and a Ter site in either the permissive or nonpermissive orientation (Figures 4B and Extended Experimental Procedures). The plasmid-based tus gene was transitorily induced only for the duration of the experiment to avoid plasmid loss. We also inserted a stretch of DNA with several spaced copies of the TTAC target tetranucleotide on both strands upstream of the Ter sites.Figure S1Experimental Systems, Related to Figure 1, Figure 2, Figure 3, Figure 4Show full caption(A) Orientation of the IS608 derivative with respect to replication direction (Related to Figure 1): Left hand cartoon shows the configuration of pBS102 in which the IS608 derivative (with the tnpA and tnpB genes replaced by a chloramphenicol resistance gene cassette) is oriented such that the active strand is part of the lagging strand template. The right hand drawing shows plasmid pBS144 in which the active IS608 strand is part of the leading strand template. Okazaki fragments on the lagging strand are indicated as short lines. The direction of DNA synthesis is indicated with half arrowheads.(B) Schematic of the excision assay (Related to Figure 2): The figure shows how excision of the inserted IS608 CmR derivative regenerates a functional beta-lactamase (bla) gene. E. coli host strains carry pAM1, in which the IS608 CmR derivative is inserted after a TTAC in the bla gene and pBS135, transposase donor plasmid with a p15A replication origin and a selectable KmR gene, under control of plac.(C) Insertions into the E. coli chromosome (Related to Figure 3): The CmR tagged IS608 derivative was carried by a temperature sensitive pSC101 plasmid, pBS156, carrying SmSp resistance. TnpA was supplied as TnpA-His in trans by plasmid pBS135. Following growth at 30°C, cells were plated on Cm plates at 43°C and then screened for the loss of the SpSm marker of the donor plasmid.(D) Insertion into Tus/Ter stalled replication forks (Related to Figure 4): Plasmid pBS167b is derived from the conjugative plasmid RP4. It carries the RP4 origin of transfer (OriTRP4) while the transfer functions are carried by the chromosome of the donor strain β2163, the origin of vegetative replication of R6K (OriVR6Kγ), a selectable SpSmR gene and the CmR IS608 derivative. The R6K replication protein, Π, is carried by the chromosome of the donor strain β2163. The recipient strain does not carry the Π gene (preventing replication of the transferred plasmid) and carries a null mutation in tus. The recipient also carries plasmid pBS135, to supply TnpA under control of plac. One of two target replicons was also present: these carried a pBR322 origin of replication, the tus gene under control of para, the araC repressor gene, a Ter sequence in the non-permissive orientation (Ternp, pBS172) or in the permissive orientation (Terp, pBS173) in front of that a stretch of several IS608 TTAC target sequences was cloned. Selection for transfer and stable maintenance of CmR in the recipient provides a plasmid population in which pBS172/pBS173 will have received IS608 insertions which can be localized by PCR.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Orientation of the IS608 derivative with respect to replication direction (Related to Figure 1): Left hand cartoon shows the configuration of pBS102 in which the IS608 derivative (with the tnpA and tnpB genes replaced by a chloramphenicol resistance gene cassette) is oriented such that the active strand is part of the lagging strand template. The right hand drawing shows plasmid pBS144 in which the active IS608 strand is part of the leading strand template. Okazaki fragments on the lagging strand are indicated as short lines. The direction of DNA synthesis is indicated with half arrowheads. (B) Schematic of the excision assay (Related to Figure 2): The figure shows how excision of the inserted IS608 CmR derivative regenerates a functional beta-lactamase (bla) gene. E. coli host strains carry pAM1, in which the IS608 CmR derivative is inserted after a TTAC in the bla gene and pBS135, transposase donor plasmid with a p15A replication origin and a selectable KmR gene, under control of plac. (C) Insertions into the E. coli chromosome (Related to Figure 3): The CmR tagged IS608 derivative was carried by a temperature sensitive pSC101 plasmid, pBS156, carrying SmSp resistance. TnpA was supplied as TnpA-His in trans by plasmid pBS135. Following growth at 30°C, cells were plated on Cm plates at 43°C and then screened for the loss of the SpSm marker of the donor plasmid. (D) Insertion into Tus/Ter stalled replication forks (Related to Figure 4): Plasmid pBS167b is derived from the conjugative plasmid RP4. It carries the RP4 origin of transfer (OriTRP4) while the transfer functions are carried by the chromosome of the donor strain β2163, the origin of vegetative replication of R6K (OriVR6Kγ), a selectable SpSmR gene and the CmR IS608 derivative. The R6K replication protein, Π, is carried by the chromosome of the donor strain β2163. The recipient strain does not carry the Π gene (preventing replication of the transferred plasmid) and carries a null mutation in tus. The recipient also carries plasmid pBS135, to supply TnpA under control of plac. One of two target replicons was also present: these carried a pBR322 origin of replication, the tus gene under control of para, the araC repressor gene, a Ter sequence in the non-permissive orientation (Ternp, pBS172) or in the permissive orientation (Terp, pBS173) in front of that a stretch of several IS608 TTAC target sequences was cloned. Selection for transfer and stable maintenance of CmR in the recipient provides a plasmid population in which pBS172/pBS173 will have received IS608 insertions which can be localized by PCR. Mapping of the IS insertion sites revealed that all occurred at TTAC sequences and were distributed over the entire length of both target plasmids, largely in an orientation expected for insertion into the lagging-strand template (Figure 4B, black arrowheads). The overall distribution was the same regardless of Ter site orientation. As for the other target plasmids used here, we confirmed that TTAC sequences were distributed roughly equally on both DNA strands (data not shown). An additional set of insertions was observed in the target plasmid carrying Ternp when compared to that with Terp (Figures 4B and 4C). To precisely map these, we used PCR analysis with primers complementary to a sequence upstream of the Ter sites and either LE (for lagging-strand insertions) or RE (for leading-strand insertions). When the target plasmid carried Ternp, amplification products from four independent experiments (Figure 4C, lanes 1–4) revealed insertions adjacent to the Ter site on the lagging-strand template (Figure 4B, filled red arrowheads; Figure 4C), although occasionally an insertion was observed on the opposite strand (lane 5). Although there was a degree of variability in the insertion distribution between experiments (compare lanes 1–4), a consistently strong signal was obtained from the TTAC site located at 63 bp from Ternp, suggesting that this is the preferred insertion site. However, insertions were also observed at the closest site, located only 26 bp from Ter (Figure 4C, lanes 1, 3, and 4). With Terp, no strong Tus-dependent insertions were observed (Figure 4D). In four independent experiments we only once isolated an insertion in this region (in the plasmid population from experiment 1, lane 1), and this had occurred into a TTAC site within the Ter sequence itself rather than immediately upstream. Thus, the lagging strand of replication forks blocked by Tus is a preferential target for IS608 insertions and these can occur in close proximity to the Tus binding site, Ter. ISDra2 is an IS200/IS605 family member from the highly radiation resistant D. radiodurans. It has a similar organization to IS608 and inserts specifically 3′ to a TTGAT pentanucleotide (Islam et al., 2003Islam S.M. Hua Y. Ohba H. Satoh K. Kikuchi M. Yanagisawa T. Narumi I. Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans.Genes Genet. Syst. 2003; 78" @default.
• W2023259875 created "2016-06-24" @default.
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• W2023259875 date "2010-08-01" @default.
• W2023259875 modified "2023-10-12" @default.
• W2023259875 title "Single-Stranded DNA Transposition Is Coupled to Host Replication" @default.
• W2023259875 cites W1508194938 @default.
• W2023259875 cites W1559827839 @default.
• W2023259875 cites W1572344748 @default.
• W2023259875 cites W1608772602 @default.
• W2023259875 cites W1974127479 @default.
• W2023259875 cites W1975495433 @default.
• W2023259875 cites W1979784608 @default.
• W2023259875 cites W1980602152 @default.
• W2023259875 cites W1980641414 @default.
• W2023259875 cites W1983622455 @default.
• W2023259875 cites W1986051089 @default.
• W2023259875 cites W1989108993 @default.
• W2023259875 cites W1993781503 @default.
• W2023259875 cites W1995677771 @default.
• W2023259875 cites W2001886199 @default.
• W2023259875 cites W2002197779 @default.
• W2023259875 cites W2003845426 @default.
• W2023259875 cites W2015316514 @default.
• W2023259875 cites W2018405580 @default.
• W2023259875 cites W2019721787 @default.
• W2023259875 cites W2023265572 @default.
• W2023259875 cites W2026966386 @default.
• W2023259875 cites W2027850822 @default.
• W2023259875 cites W2032283008 @default.
• W2023259875 cites W2033889514 @default.
• W2023259875 cites W2036099589 @default.
• W2023259875 cites W2040269512 @default.
• W2023259875 cites W2050228332 @default.
• W2023259875 cites W2050630486 @default.
• W2023259875 cites W2050878024 @default.
• W2023259875 cites W2051449352 @default.
• W2023259875 cites W2052561020 @default.
• W2023259875 cites W2061411740 @default.
• W2023259875 cites W2066417696 @default.
• W2023259875 cites W2075864467 @default.
• W2023259875 cites W2076387647 @default.
• W2023259875 cites W2076712367 @default.
• W2023259875 cites W2091355621 @default.
• W2023259875 cites W2093848749 @default.
• W2023259875 cites W2105474110 @default.
• W2023259875 cites W2109550125 @default.
• W2023259875 cites W2111356152 @default.
• W2023259875 cites W2113988248 @default.
• W2023259875 cites W2119344497 @default.
• W2023259875 cites W2121467305 @default.
• W2023259875 cites W2129717376 @default.
• W2023259875 cites W2134727169 @default.
• W2023259875 cites W2137874473 @default.
• W2023259875 cites W2139275024 @default.
• W2023259875 cites W2143804506 @default.
• W2023259875 cites W2157358322 @default.
• W2023259875 cites W2163827697 @default.
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