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Tipping the balance between replicative and simple transposition

2001; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês

10.1093/emboj/20.11.2923

1460-2075

Norma P. Tavakoli,

Advanced biosensing and bioanalysis techniques

Article1 June 2001free access Tipping the balance between replicative and simple transposition Norma P. Tavakoli Norma P. Tavakoli Division of Infectious Disease, Wadsworth Center, New York State Department of Health, State University of New York at Albany, Albany, NY, 12201-2002 USA Search for more papers by this author Keith M. Derbyshire Corresponding Author Keith M. Derbyshire Division of Infectious Disease, Wadsworth Center, New York State Department of Health, State University of New York at Albany, Albany, NY, 12201-2002 USA Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY, 12201-2002 USA David Axelrod Institute, NYS Department of Health, PO Box 22002, Albany, NY, 12201-2002 USA Search for more papers by this author Norma P. Tavakoli Norma P. Tavakoli Division of Infectious Disease, Wadsworth Center, New York State Department of Health, State University of New York at Albany, Albany, NY, 12201-2002 USA Search for more papers by this author Keith M. Derbyshire Corresponding Author Keith M. Derbyshire Division of Infectious Disease, Wadsworth Center, New York State Department of Health, State University of New York at Albany, Albany, NY, 12201-2002 USA Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY, 12201-2002 USA David Axelrod Institute, NYS Department of Health, PO Box 22002, Albany, NY, 12201-2002 USA Search for more papers by this author Author Information Norma P. Tavakoli1 and Keith M. Derbyshire 1,2,3 1Division of Infectious Disease, Wadsworth Center, New York State Department of Health, State University of New York at Albany, Albany, NY, 12201-2002 USA 2Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY, 12201-2002 USA 3David Axelrod Institute, NYS Department of Health, PO Box 22002, Albany, NY, 12201-2002 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2923-2930https://doi.org/10.1093/emboj/20.11.2923 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The bacterial insertion sequence IS903 has the unusual ability to transpose both replicatively and non-replicatively. The majority of products are simple insertions, while co-integrates, the product of replicative transposition, occur at a low frequency (<0.1% of simple insertions). In order to define the critical steps that determine the outcome of IS903 transposition, we have isolated mutants that specifically increase the rate of replicative transposition. Here we show that the nucleotide immediately flanking the transposon influences both overall transposition frequency and co-integrate formation. In particular, when the 3′-flanking nucleotide is A, co-integrates are increased 500-fold compared with a 3′ C. In addition, we have isolated five transposase mutants that increase replicative transposition. These residues are close to the catalytic residues and are thus likely to be part of the active site. These are the first transposase mutations described that affect the product of transposition. Our results are consistent with the hypothesis that a delay in cleavage of the 5′-flanking DNA will increase the effective half-life of the 3′-nicked transposon intermediate and consequently enhance co-integrate formation. Introduction Bacterial transposons move from one genomic location to another by a process that is independent of DNA homology. These transposons move by one of two pathways, non-replicative (also called simple insertion, conservative and cut-and-paste) and replicative (Figure 1a and b), to generate transposition products called simple insertions or co-integrates, respectively (Mizuuchi, 1992; Craig, 1996; Haren et al., 1999). Most transposons move exclusively by one pathway or the other. IS903 and IS1 are unusual as they have been shown to carry out both simple insertion and replicative transposition (Ohtsubo et al., 1981; Weinert et al., 1984; Turlan and Chandler, 1995). Figure 1.Alternate pathways of transposition for IS903. (a) Simple insertion of IS903 from pKD498 into the conjugative plasmid, pOX38. These transposition events can be detected by transfer of chloramphenicol resistance (Cmr) into a suitable recipient using a mating-out assay. (b) Replicative transposition results in a co-integrate structure that fuses donor and target replicons via two copies of the transposon. Co-integrates can be identified by co-transfer of both Cmr and kanamycin resistance (Kmr) by pOX38. Note that co-integrates may also form following simple transposition from a plasmid dimer, but that in all these experiments propagation of plasmid DNAs in recA− strains precluded dimer formation. The transposon on pKD498 is indicated by the heavy line and the inverted repeats are indicated by triangles. Flanking donor DNA and target DNA are indicated by thin lines. Download figure Download PowerPoint Although the products of non-replicative and replicative transposition look very different, extensive in vitro studies have confirmed models based on in vivo experiments to show that the biochemical steps in both pathways are remarkably similar (see below and reviews by Mizuuchi, 1997; Haren et al., 1999). The major distinguishing feature between the two pathways is whether a double-strand break or a single-strand nick occurs at the ends of the transposon before integration (Turlan and Chandler, 2000). A double-strand break effectively removes all connections with the donor DNA and thus precludes co-integrate formation. In contrast, a nick allows maintenance of connections to both donor and target replicons after integration, and, following replication, results in formation of a co-integrate. The initial step of both transposition pathways involves the generation of a 3′ OH at the transposon termini (Figure 2a). In the simple insertion pathway, subsequent cleavage of the 5′-flanking DNA generates an excised transposon (Figure 2b). The 3′ OH ends of the excised transposon then act as nucleophiles in a concerted strand transfer reaction that results in integration of the transposon into a target (Figure 2d). The target DNA is cleaved in a staggered manner and the short single-stranded regions that flank the newly inserted transposon are repaired by the host replication apparatus to generate short direct repeats, a signature of the transposition reaction. The mechanism of cleavage of the 5′-flanking strand varies. For IS10 and IS50, the excised transposon is generated via a hairpin intermediate (Kennedy et al., 1998; Bhasin et al., 1999). The hairpin is formed when the 3′ OH at each transposon end attacks the phosphodiester backbone at the 5′ ends of the transposon. This transesterification not only results in the joining of the 3′ OH to the 5′ end of the transposon, but also releases flanking donor DNA. The hairpin is resolved by a second, transposase-mediated nick (hydrolysis) to generate the excised transposon intermediate with 3′ OH termini. Tn7 also generates double-strand breaks at each transposon end, but by a different mechanism. In contrast to IS10 and IS50, Tn7 utilizes two Tn7-encoded proteins to process each flanking strand (Sarnovsky et al., 1996). TnsB, a transposase-related protein, nicks the 3′ ends of the transposon. TnsA, a site-specific endonuclease, cleaves the 5′ ends to generate an excised transposon, which is then inserted into a target (May and Craig, 1996; Hickman et al., 2000). Figure 2.Summary of the biochemical steps that occur in simple and replicative transposition pathways. Nicking at the 3′ ends is the initial step in both pathways (a). In the simple insertion pathway, this is followed by cleavage of the 5′-flanking DNA (b), which generates an excised transposon. Interaction with a target (c) allows strand transfer to occur, which results in a simple insertion (d). Replicative transposition (a, e and f) occurs via a strand transfer reaction (e) involving the nicked transposon and a target to generate a strand transfer intermediate. Replication of this intermediate using the exposed 3′ OHs in the target DNA as priming sites (f) results in duplication of the transposon and a co-integrate structure. IRs are represented by filled triangles, the transposon DNA is shown as a thick line, flanking and target DNAs as thin lines, and cleavage sites by small vertical arrows. Download figure Download PowerPoint In the replicative pathway (Figure 2e and f), as characterized for bacteriophage Mu, second strand cleavage does not occur. Instead, strand transfer results in fusion of target and donor DNAs to form a strand transfer intermediate, which consists of a copy of the transposon joined to both donor and target DNA (Figure 2e) (Craigie and Mizuuchi, 1985; Naigamwalla and Chaconas, 1997). The exposed 3′ OHs of the target in this intermediate are used to prime DNA synthesis, allowing replication of the entire transposon to form a co-integrate structure (Figure 2f). The co-integrate is composed of the donor and target replicons fused by two copies of the transposable element in direct orientation (Figure 1b). To understand the unique properties of IS903 that allow it to move by both replicative and non-replicative pathways, we have isolated both transposase and DNA mutants that increase the frequency of replicative transposition. IS903 is an insertion sequence of 1057 bp that has inverted repeats (IRs) of 18 bp and encodes a transposase of 307 amino acids (Grindley and Joyce, 1981). The IS903 transposase contains the highly conserved DDE and YREK motifs common to the IS4 family of transposons, which are thought to play a key role in catalysis (Rezsohazy et al., 1993; Tavakoli et al., 1997). The 18 bp IR has been subdivided into two functional regions based on genetic and biochemical analyses (Derbyshire et al., 1987; Derbyshire and Grindley, 1992). The inner 12 bp (bp 6–18) are essential for binding of transposase, which makes both major and minor groove contacts to this region. The outer domain (bp 1–3) is involved in a step subsequent to binding, probably catalysis, since this is the site of DNA cleavage and is thus likely to be an intimate part of the transposase active site. This is also consistent with the phenotype of mutations in the outer domain, which considerably reduce transposition frequency but do not affect transposase binding (Derbyshire et al., 1987; Derbyshire and Grindley, 1992). Furthermore, mutations at the transposon termini affect the outcome of the transposition reaction (Tavakoli and Derbyshire, 1999). Substitution of the wild-type C at the transposon termini reduces transposition frequency by 2–4 orders of magnitude, but increases co-integrate formation to 30–50%. This suggested that the nucleotides at the transposon termini affect both first and second strand processing, and prompted us to investigate the effect of other DNA and protein mutations on co-integrate formation in IS903 transposition. Results Effect of flanking DNA on transposition and co-integrate formation Point mutations at the termini of the IR decrease overall transposition frequency and increase co-integrate formation, suggesting that the terminal nucleotide plays a role in the transposon excision process (Tavakoli and Derbyshire, 1999). We hypothesized that the nucleotide immediately flanking the IR might also affect the reaction, as transposase mediates cleavage of the phosphodiester backbone at the transposon–flank junction. Four transposon vectors were made that differed only by the nucleotide flanking both transposon ends. Mating-out assays were performed to determine the effect on transposition and co-integrate formation (Table I). Table 1. Effect of flanking DNA on co-integrate formation IS903 inverted repeat +1 −1 −2 −3 Transposase Flanking DNA (−1 −2 −3) Transposition frequency % co-integrates w/t CTG 1.0 × 10−3 0.01 w/t TTG 1.8 × 10−3 0.35 w/t ATG 3.1 × 10−3 5.4 w/t GTG 2.0 × 10−4 <0.02 V119A CTG 1.9 × 10−2 0.01 V119A TTG 8.2 × 10−3 0.35 V119A ATG 1.3 × 10−2 4.1 V119A GTG 5.5 × 10−3 0.15 w/t ACG 3.0 × 10−3 4.2 w/t ATG 3.1 × 10−3 5.4 w/t AAG 3.0 × 10−3 1.7 w/t AGG 2.9 × 10−3 13.7 w/t AGC 2.2 × 10−3 6.6 w/t AGT 3.0 × 10−3 6.2 w/t AGA 3.0 × 10−3 1.6 w/t AGG 3.0 × 10−3 13.7 The sequence of the 18 bp IS903 IR with flanking DNA (−1, −2 and −3) is shown at the top of the table. The flanking nucleotide altered for each assay is indicated in bold and underlined. w/t, wild-type transposase. V119A, transposase with a valine to alanine substitution at amino acid 119. Transposition frequencies are the average of at least six experiments. Transposition frequencies were high, as expected for a wild-type transposon but, most remarkably, a 3′-flanking A increased co-integrate formation to 5% (500-fold higher than a C). To determine whether this effect required an A at both flanks, derivatives were made in which the transposon was flanked by a 3′ C and a 3′ A. The co-integrate levels were reduced to levels similar to those of a construct with C at both flanking sequences (data not shown). A 3′-flanking C at one transposon end, therefore, has a dominant effect over an A at the other, suggesting that a double-strand break is made efficiently at the C nucleotide end, which commits the transposon to the simple insertion pathway. We note that a G nucleotide flanking the 3′ ends of the transposon (G at −1) consistently lowered the transposition frequency (5-fold compared with other flanking nucleotides). We have observed this subtle reduction in transposition frequency with other transposon constructs flanked by a 3′ G (see Table IV and data not shown). To demonstrate further that these mutations had increased the frequency of replicative transposition, we utilized a hyperactive transposase mutant (V119A) to increase the overall transposition frequency (Tavakoli and Derbyshire, 1999). This mutation is just two amino acids downstream from the first aspartate of the catalytic DDE motif (Figure 3) and increases transposition frequency ∼20-fold. Transposons that incorporated the transposase mutant V119A together with each possible flanking nucleotide were constructed, and co-integrate formation was measured (Table I). As expected, the transposition frequency of each derivative containing the V119A substitution was increased. Most importantly, the high level of co-integrate formation with a flanking 3′ A was maintained at a level similar to that mediated by wild-type transposase. These results, combined with the effect we had observed previously for alterations at the transposon termini, show that the dinucleotide at the transposon–donor junction can have a dramatic effect on the outcome of transposition. Figure 3.Alignment of amino acids around the conserved active site residues of IS903 and IS10 transposases. The DDE (bold) and YREK motifs are shown in the center line and aligned with the conserved residues in the IS903 and IS10 transposases (boxed). The IS903 residues considered candidates for involvement in strand cleavage and transfer (affecting co-integrate formation in IS903) are shown above the alignment. Amino acid substitutions discussed in more detail in the text are indicated. The IS10 mutants discussed in the text, and their phenotypes, are indicated below the alignment. The vertical dots align non-conserved residues that were mutated. Download figure Download PowerPoint Nucleotides at positions −2 and −3 have a minor effect on co-integrate formation To extend the analysis of the flanking DNA sequence, nucleotides at positions −2 and −3 were varied. Since the possible number of combinations of nucleotides is high, we limited the combinations to nucleotides that showed the highest percentage of co-integrate formation. Accordingly, constructs were made that retained A at −1, which allowed for the detection of both positive and negative effects on replicative transposition. Only small changes were observed in the level of co-integrate formation when varying the nucleotide at −2 (Table I). The highest level observed was with G at nucleotide −2 (13.7%). Four additional constructs were tested that had A and G at positions −1 and −2, but varied the nucleotides at −3 (Table I). Co-integrate formation varied from 1.6 to 13.7%. These results confirm that the nucleotide at position −1 has the most dramatic effect on the outcome of the transposition reaction (500-fold for a 3′ A) and that nucleotides further away play only a minor role in modifying the outcome of transposition. Transposase mutants that affect co-integrate formation The phenotypes of the DNA mutants were consistent with the hypothesis that they were delaying the cleavage of the flanking DNA, thereby increasing the effective half-life of the nicked transposon intermediate (Figure 2a) and the chance of target capture to form a strand transfer intermediate (Figure 2e). Based on this hypothesis, we predicted that any transposase mutant that prolonged the half-life of the nicked transposon substrate should also elevate replicative transposition. To identify candidate residues, we considered a collection of IS10 transposase mutants that were defective in transposon excision and strand transfer. We focused on five mutants that showed defects in second strand cleavage and strand transfer (W98R, I101S), hairpin formation (P167S) or exhibited a hypernicking phenotype (A162T and M289I) (Haniford et al., 1989; Bolland and Kleckner, 1995; Kennedy and Haniford, 1996; Kennedy et al., 1998). In particular, the phenotype of the latter three mutants would be predicted to increase the effective half-life of the nicked intermediate and thus enhance co-integrate formation, although this had not been tested for IS10. By aligning the IS903 and IS10 transposase sequences, we identified the equivalent residues in the IS903 transposase (Figure 3; S122, L125, G194, R199 and S256). Although these residues are not conserved, they are located in close proximity to the highly conserved active site residues [in the case of IS903, D121, D193 and E259 (Tavakoli et al., 1997); and for IS10, D97, D161 and E292 (Bolland and Kleckner, 1996)] (Figure 3). Site-specific mutagenesis of candidate residues was used to introduce the desired mutations. Mutant transposases were cloned into a transposon donor vector and assayed by in vivo mating-out assays. The overall transposition frequencies and levels of co-integrate formation are shown in Table II. Table 2. Transposition frequency and level of co-integrate formation of IS903 mutants IS903 Tnp residue Amino acid change Transposition frequency % co-integrates Equivalent IS10 Tnp residue w/t 1.0 × 10−3 0.01 S122 A 2.8 × 10−6 – W98R R 1.0 × 10−4 – (strand transfer defect) G <1.0 × 10−8 – L125 A <1.0 × 10−8 – I101S S <1.0 × 10−8 – (strand transfer defect) G194 S 1.5 × 10−4 – A162T A 4.0 × 10−6 2 (hypernicking mutant) T 2.0 × 10−6 – I <1.0 × 10−8 – R <1.0 × 10−8 – N <1.0 × 10−8 – R199 A 3.9 × 10−7 – P167S S <1.0 × 10−8 – (hairpin defect) P <1.0 × 10−8 – S256 M 8.5 × 10−6 30 M289I A 5.0 × 10−6 2 (hypernicking mutant) V 2.1 × 10−6 1 T 3.8 × 10−7 10 D <1.0 × 10−8 – E <1.0 × 10−8 – K <1.0 × 10−8 – G <1.0 × 10−8 – R <1.0 × 10−8 – I <1.0 × 10−8 – Co-integrates are indicated as a percentage of the transposition events and are an average of at least six assays. Equivalent IS10 transposase (Tnp) mutants and their relevant phenotypes are also listed (see text). A dash (–) indicates that no co-integrates were observed or that the transposition frequency was too low to allow detection of co-integrates. Substitutions at S122 and L125 reduced transposition to undetectable levels or, when transposition was detected, the transconjugants resulted from simple insertions and were not studied further. In contrast, substitutions at residues G194 and S256 increased co-integrate formation. Five mutant transposases G194A, S256M, S256A, S256V and S256T, showed an increase in co-integrate formation when compared with wild-type transposase. Substitutions of G194 to S and T reduced the transposition frequency but did not affect co-integrate formation, while substitutions to I, R and N reduced transposition to undetectable levels. Other substitutions of S256 to D, E, K, G, R and I also abolished transposition. Two mutants, G194A and S256M, were characterized further as they generated co-integrates at the highest frequency. Transposition mediated by each mutant transposase was reduced by 2–3 orders of magnitude compared with wild-type, and so we considered the possibility that the elevated formation of co-integrates might simply reflect a reduction in simple transposition events. Double mutants were made with the hyperactive transposase mutant V119A in order to rescue transposition. The V119A mutation rescued the transposition frequency of each mutant but maintained the same level of co-integrate formation (Table III). This demonstrates that the amino acid substitutions are specifically increasing replicative transposition. A double mutant containing both G194A and S256M mutations showed no additional increase in co-integrate formation above the value obtained with S256M (Table III), indicating that the mutations are probably acting in the same way. Table 3. Transposition rescue by the hyperactive transposase mutant V119A does not alter co-integrate formation IS903 Tnp Transposition frequency % co-integrates w/t 1.0 × 10−3 0.01 V119A 1.9 × 10−2 0.01 G194A 4.0 × 10−6 2.0 G194A + V119A 1.5 × 10−2 1.0 S256M 8.5 × 10−6 30.0 S256M + V119A 5.2 × 10−2 16.0 G194A + S256M 2.7 × 10−6 30.0 R199A 3.9 × 10−7 – R199A + V119A 1.3 × 10−3 0.5 R199S <1.0 × 10−8 – R199S + V119A 2.1 × 10−4 0.3 R199P <1.0 × 10−8 – R199P + V119A <1.0 × 10−8 – Y252A 8.0 × 10−7 – Y252A + V119A 5.5 × 10−4 0.5 R255A 4.0 × 10−8 – R255A + V119A 7.6 × 10−5 9.0 K266A <1.0 × 10−8 – K266A + V119A 5.6 × 10−5 <0.05 K266G <1.0 × 10−8 – K266G + V119A 4.4 × 10−4 <0.01 A dash (–) indicates that no co-integrates were observed or that the transposition frequency was too low to allow detection of co-integrates. Table 4. Combination of transposase mutants with all four possible flanking nucleotides Transposase Flanking nucleotide (−1) Transposition frequency % co-integrates w/t C 1.0 × 10−3 0.01 w/t T 1.8 × 10−3 0.35 w/t A 3.1 × 10−3 5.4 w/t G 2.0 × 10−4 <0.02 V119A C 1.9 × 10−2 0.01 V119A T 8.2 × 10−3 0.35 V119A A 1.3 × 10−2 4.1 V119A G 5.5 × 10−3 0.15 G194A C 4.0 × 10−6 2.0 G194A + V119A C 2.8 × 10−3 1.5 G194A + V119A T 1.7 × 10−3 2.1 G194A + V119A A 1.3 × 10−3 13.0 G194A + V119A G 3.2 × 10−4 6.0 S256M C 8.5 × 10−6 30.0 S256M + V119A C 2.7 × 10−3 15.0 S256M + V119A T 4.8 × 10−4 13.0 S256M + V119A A 2.9 × 10−3 23.0 S256M + V119A G 4.8 × 10−4 14.0 K266G C <1.0 × 10−8 – K266G + V119A C 4.4 × 10−4 <0.01 K266G + V119A T 1.0 × 10−6 – K266G + V119A A 5.2 × 10−4 7.0 K266G + V119A G 8.0 × 10−6 – A dash (–) indicates that no co-integrates were observed or the transposition frequency was too low to allow detection of co-integrates. Data for wild-type and V119A transposases are taken from Table I. The ability of V119A to rescue transposition of the mutant transposases prompted us to re-examine the IS903 R199 substitutions (nominally equivalent to P167 in IS10), which exhibited barely detectable levels of transposition and thus prevented detection of co-integrates (Table II). In vitro studies have shown that P167S is defective in hairpin formation, but nicking occurs efficiently at the 3′ ends of the transposon (Kennedy et al., 1998). Double mutants were therefore constructed with the V119A mutation in order to suppress the transposition defect of the R199 substitutions. Transposition frequency was rescued for two of the double mutants but not for the R199P transposase. More importantly, a small but reproducible increase (30- to 50-fold compared with wild-type transposase) in co-integrate formation was observed for the R199A and R199S double mutants (Table III). In an effort to identify other transposase mutations that increased replicative transposition, we screened substitutions of the highly conserved YREK motif (Figure 3). In agreement with previous results (Haniford et al., 1989; Bolland and Kleckner, 1996; Tavakoli et al., 1997; Davies et al., 2000), substitution of these residues had a very deleterious affect on transposition (Table III). Transposition of these mutant transposases could be rescued by introducing the hyperactive V119A mutation, and for two of these derivatives, Y252A and R255A, significant levels of co-integrates were detected (Table III). Several substitutions of K266 were examined (R, G and A), but none increased the level of co-integrate formation above background levels (Table III and data not shown). Hypernicking and hairpin defect mutants of IS10 do not allow co-integrate formation We also examined the effect of the IS10 mutations on IS10 transposition, as these had only been examined under in vitro conditions that would not have allowed detection of replicative transposition intermediates (Bolland and Kleckner, 1995). Transposition of IS10 was monitored using derivatives that allowed direct selection for co-integrates. No co-integrates were detected with either the wild-type IS10 transposase (<1 in 105 events) or any of the mutants tested (A162T, P167S and M289I; data not shown). These results are in contrast to those with the wild-type IS903 and mutants G194A, R199A and S256M, which generate co-integrates, and thus imply that there is a fundamental difference in the mechanism of transposition between the two elements (see Discussion). Combining transposase mutants and dinucleotide pairs We examined whether the protein and DNA mutants are additive in their effect on replicative transposition. Both G194A and S256M (combined with V119A) transposases were introduced into transposon vectors that were flanked by different nucleotides. In each case, the G194A and S256M transposases increased the co-integrate frequency (Table IV), but a flanking A did not significantly increase the co-integrate levels above those observed with other flanking nucleotides. This suggests that the effect of both the protein and DNA substitutions is at a similar step in the replicative transposition pathway. A K266G substitution does not increase replicative transposition, but is sensitive to the flanking nucleotide A substitution of glycine for lysine at transposase residue 266 exhibited an unusual sensitivity to the nature of the flanking DNA. Transposition mediated by K266G transposase was almost undetectable, but could be rescued significantly by introducing the V119A substitution (Table IV). However, the efficiency with which the V119A substitution rescued transposition mediated by transposase K266G was very dependent on the flanking nucleotide. Transposition was significantly higher when flanked by a 3′ A or C compared with a G or T (520-fold comparing a flanking 3′ A with a 3′ T; Table IV). This is in contrast to the effect of the flanking nucleotide on transposition mediated by the wild-type, V119A and several other mutant transposases that we have screened. In these latter cases, a 5-fold reduction in transposition frequency is observed when the transposon is flanked by a 3′ G, but for other flanking nucleotides there was little, or no variation in transposition. Discussion IS903 is unusual among bacterial transposable elements in that it has the ability to transpose by two pathways, which generate different products: simple insertions or co-integrates. Here, we have described a series of DNA and protein mutants that specifically increase the frequency of replicative transposition by IS903. These results can be rationalized in light of current models of cut-and-paste versus replicative transposition (reviewed in Craig, 1996; Turlan and Chandler, 2000), and also provide an explanation for the ability of IS903 to move by both pathways. The predominant product of IS903 transposition is a simple insertion. We propose that normally IS903 rapidly processes the flanking DNA to generate an excised transposon resulting in a simple insertion. The mutants we have described would delay the processing of the 5′-flanking DNA at both ends of the transposon. This would effectively increase the half-life of the nicked product (Figure 2a), and thereby increase the chance of target capture to form a strand transfer intermediate (Figure 2e) that is then replicated to generate a co-integrate. The low level of co-integrates that can be detected with the wild-type transposon is most likely to be due to the effect of the 3′-flanking nucleotide. As observed in Table I, this can have a 500-fold effect on the frequency of co-integrate formation (from 0.01 to 5% depending on whether the transposon is flanked by a C or an A) even with a wild-type transposon. To our knowledge, this is the first time that substitutions in the flanking DNA have been documented to affect the outcome of transposition. A considerable amount of biochemical evidence, from work with a number of transposons, has suggested that the distinguishing feature between simple insertion and replicative transposition is whether a double-strand break, or a nick, is made at the ends of the transposon before target capture (Craig, 1996; Turlan and Chandler, 2000). This was confirmed by a combination of in vivo and in vitro experiments with Tn7 (May a