Portal de Periódicos da CAPES

Accessory factors determine the order of strand exchange in Xer recombination at psi

2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês

10.1093/emboj/cdf379

1460-2075

Migena Bregu,

DNA Repair Mechanisms

Article15 July 2002free access Accessory factors determine the order of strand exchange in Xer recombination at psi Migena Bregu Migena Bregu Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author David J. Sherratt David J. Sherratt Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Sean D. Colloms Corresponding Author Sean D. Colloms Present address: Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author Migena Bregu Migena Bregu Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author David J. Sherratt David J. Sherratt Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK Search for more papers by this author Sean D. Colloms Corresponding Author Sean D. Colloms Present address: Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow, G11 6NU UK Search for more papers by this author Author Information Migena Bregu1, David J. Sherratt1 and Sean D. Colloms 2 1Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU UK 2Present address: Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow, G11 6NU UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3888-3897https://doi.org/10.1093/emboj/cdf379 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Xer site-specific recombination in Escherichia coli converts plasmid multimers to monomers, thereby ensuring their correct segregation at cell division. Xer recombination at the psi site of plasmid pSC101 is preferentially intramolecular, giving products of a single topology. This intramolecular selectivity is imposed by accessory proteins, which bind at psi accessory sequences and activate Xer recombination at the psi core. Strand exchange proceeds sequentially within the psi core; XerC first exchanges top strands to produce Holliday junctions, then XerD exchanges bottom strands to give final products. In this study, recombination was analysed at sites in which the psi core was inverted with respect to the accessory sequences. A plasmid containing two inverted-core psi sites recombined with a reversed order of strand exchange, but with unchanged product topology. Thus the architecture of the synapse, formed by accessory proteins binding to accessory sequences, determines the order of strand exchange at psi. This finding has important implications for the way in which accessory proteins interact with the recombinases. Introduction Site-specific recombination systems are implicated in a variety of DNA rearrangements in microorganisms (Hallet and Sherratt, 1997). Recombination is catalysed by recombinase proteins that carry out DNA strand cleavage and transfer reactions at short DNA sequences known as recombination core sites (Stark et al., 1992). Site-specific recombination often requires additional accessory proteins, which bind to accessory sequences adjacent to the recombination core. The recombinase and accessory proteins form a highly organized protein–DNA complex with the recombination site DNA, and together exert control over the efficiency and timing of the recombination reaction. The way in which accessory proteins bound at accessory sequences on a DNA molecule can regulate and control strand exchange reactions by recombinases is the subject of this study. The Xer site-specific recombination system of Escherichia coli resolves multimeric forms of circular replicons to allow efficient segregation of monomers to daughter cells at cell division (Sherratt et al., 1995). Two related recombinases, XerC and XerD, act at the chromosomal site dif and at plasmid-borne sites such as cer of ColE1 and psi of pSC101 (Colloms et al., 1990; Blakely et al., 1993; Cornet et al., 1994). Xer recombination sites share an ∼30 bp core sequence, containing an 11 bp XerC-binding site and an 11 bp XerD-binding site separated by a 6–8 bp asymmetric central region. In addition, recombination at cer and psi requires accessory sequences and accessory proteins, which ensure that recombination is preferentially intramolecular, converting plasmid multimers to monomers and not vice versa (Stirling et al., 1989; Summers, 1989). PepA, an aminopeptidase with DNA-binding activity, and ArgR, the arginine repressor, bind to ∼180 bp of accessory sequences adjacent to the cer core and are both required for recombination at cer (Colloms et al., 1996; Alén et al., 1997). Similarly, PepA and ArcA, an anaerobic response regulator protein, act at ∼160 bp of accessory sequences adjacent to the psi core (Colloms et al., 1996, 1998). Xer site-specific recombination at psi has been reconstituted in vitro with purified PepA, XerC and XerD proteins. Although ArcA is essential for efficient recombination in vivo, it is not required in vitro (Colloms et al., 1998). Recombination on a substrate containing two psi sites in direct repeat generates two product rings, which are interlinked to form a specific four-noded catenane (Figure 1A; Colloms et al., 1997). Prior to strand exchange at psi, accessory proteins bind to two recombination sites and assemble a complex in which the DNA accessory sequences of both sites are wrapped a precise number of times around the accessory proteins (Colloms et al., 1997; Bath et al., 1999). This productive synaptic complex can only be formed easily between directly repeated sites on the same DNA molecule, and the recombinases only act efficiently at cer and psi cores within this complex. The requirement for this complex acts as a checkpoint (or topological filter) that favours resolution over inversion and intermolecular recombination. Because recombination occurs by a defined pathway, the products have a defined topology. Products of defined topology are produced by other systems that use accessory sequences and proteins to trap a fixed number of supercoils between recombining sites prior to recombination (e.g. Tn3/γδ resolvase and the DNA invertases; reviewed in Stark and Boocock, 1995). In other site-specific recombination reactions (such as those catalysed by λ integrase), recombination occurs after random collision of sites, trapping a variable number of supercoils between recombining sites. Such systems give mixtures of free circles and catenanes with even numbers of crossings from substrates with directly repeated sites, and knots with odd numbers of crossings from substrates with sites in inverted repeat (Figure 1B). Figure 1.Topology of site-specific recombination reactions. (A) Xer recombination at psi occurs only after the formation of a synaptic complex with a defined local structure (shown boxed). This productive synapse, formed by wrapping the accessory sequences of two recombination sites around the accessory protein PepA, traps a specific number of topological nodes. Strand exchange occurs by a defined mechanism and the product is a right-handed four-noded catenane with antiparallel psi sites. (B) Many site-specific recombination systems display no topological specificity. A random number of supercoils are trapped when the recombination sites come together, and recombination generates products of mixed topology. Sites in direct repeat yield two circles, which are either unlinked, or linked in catenanes with even numbers of topological crossings or nodes, as shown. Sites in an inverted repeat yield single circular products, which are either unknotted or knotted with an odd number of nodes. Knots and catenanes produced in these reactions are generally members of the torus family of knots and catenanes, as shown. Download figure Download PowerPoint Xer recombination occurs by a pair of sequential single-strand exchanges, which first form and then resolve a Holliday junction (HJ) intermediate. At psi, these strand exchanges proceed with a strictly defined order: XerC first exchanges top strands and then XerD exchanges bottom strands. Xer cleavage and strand exchange at dif also proceed with a preferred order (Blakely et al., 1997; Neilson et al., 1999). The order of strand exchange at dif is determined by the sequence of the core site and its interactions with the recombinases (Hallet et al., 1999; Blakely et al., 2000). In this study, we show that the order of strand exchange at psi is dictated by the architecture of the synaptic complex formed by accessory proteins and sequences, so that on an appropriate DNA substrate the normal order of strand exchange is reversed. The cell division protein FtsK also alters the order of Xer strand exchange at dif (Aussel et al., 2002), so it seems likely that the results presented here will have implications for the mode of action of FtsK at dif. Results Efficient recombination at psi requires accessory sequences To investigate whether accessory sequences are absolutely required for recombination and topological selectivity at psi, we constructed a family of plasmids containing psi core sites with and without accessory sequences in different combinations. The ability of these plasmids to undergo recombination was tested in vitro using purified XerC and XerD in the presence and absence of PepA. Reactions were carried out under normal conditions (with 10% glycerol in the reaction buffer) and also in the presence of 40% glycerol, which has been shown previously to allow unusual Xer reactions to take place (Cornet et al., 1997; Hallet et al., 1999). Recombination between two full psi sites in the presence of PepA gave a four-noded catenane in both conditions, but 40% glycerol inhibited recombination and increased the yield of HJ intermediates (Figure 2). In the absence of PepA, no recombination was observed at 10% glycerol. However, a small amount of PepA-independent recombination was observed at 40% glycerol, giving products of mixed topology (four-, six- and eight-noded catenanes were observed). Figure 2.Recombination at psi sites with and without accessory sequences. (A) Maps of p-psi.psi, containing two psi sites in direct repeat, and p-CD.DC, containing two psi cores without accessory sequences in inverted repeat. Accessory sequences are shown as thick black lines; XerC- and XerD-binding sites in the psi core are shown as filled and open triangles, respectively. (B) Topological analysis of recombination products. Plasmids were reacted with XerC and XerD with or without PepA, with either 10 or 40% glycerol as indicated. Reactions were nicked and run on a 0.7% agarose gel. Bands are indicated as follows: oc S, open circle substrate; lin S, linear substrate; sc S, supercoiled substrate; oc 4-cat, four-noded catenated product nicked on both circles; 1/2 nicked 4-cat, four-noded catenated product nicked on the large circle but still supercoiled on the small circle; sc 4-cat, fully supercoiled four-noded catenane; 3-knot, 5-knot and 7-knot, nicked knotted inversion products with three, five and seven nodes, respectively; ∞HJ, HJ intermediate nicked at the HJ on the recombinant strand, with consequent loss of any knotting or catenation. Download figure Download PowerPoint A plasmid with one full psi site in direct repeat with a psi core (p-psi.CD), as well as plasmids containing two psi cores in direct (p-CD.CD) or inverted (p-CD.DC) repeat, did not recombine efficiently in any of the conditions tested. The efficiency and topology of recombination on these substrates were not affected by the addition of PepA (Figure 2; data not shown). A low level of recombination was observed at 40% glycerol, yielding products of mixed topology (Figure 2; data not shown). These results demonstrate that two sets of accessory sequences together with PepA are required for efficient recombination at psi. In the absence of PepA and/or accessory sequences, very low levels of recombination occur only at 40% glycerol, giving products of mixed topology. Recombination at the inverted-core psiDC site Wild-type psi consists of a 28 bp core, comprising 11 bp XerC- and XerD-binding sites separated by a 6 bp asymmetric central region (Figure 3A), with ∼160 bp of accessory sequences adjacent to the XerC-binding site. Xer cleavage and strand exchange take place within the psi core, on either side of the 6 bp central region. To investigate how accessory sequences affect alignment of recombination cores and subsequent activation of strand exchange, we constructed psiDC, in which the entire psi core has been inverted with respect to the accessory sequences (Figure 3B). Therefore, psiDC has an 11 bp XerD-binding site adjacent to the accessory sequences and an 11 bp XerC-binding site distal to the accessory sequences. Figure 3.Recombination at psiDC. (A) Sequence of the psi core showing XerC- and XerD-binding and cleavage sites. (B) Diagram of the psi and psiDC sites, showing accessory sequences, to which PepA binds, and the core site with XerC- and XerD-binding sites represented as filled and open triangles, respectively. (C) Diagrams of p-psiDC.psi and p-psiDC.psiDC and their major recombination products. Plasmids were reacted with XerC and XerD in the presence or absence of PepA, with 10 or 40% glycerol (as indicated). Reactions were nicked and run on a 0.7% agarose gel. Bands are indicated as in Figure 2. Download figure Download PowerPoint The psiDC site was used to construct two recombination substrates (Figure 3C). p-psiDC.psi contains one wild-type psi site and one psiDC site. The accessory sequences of the two sites are directly repeated in this plasmid, but the core sites are in inverted repeat. Therefore, Xer recombination on p-psiDC.psi is expected to yield a single circular product, with the segment between the two cores inverted. The plasmid p-psiDC.psiDC contains two psiDC sites in direct repeat. The accessory sequences and the cores of the two sites in p-psiDC.psiDC are directly repeated. There fore, Xer recombination is expected to yield two circular resolution products, which may be catenated. Both plasmids were incubated with XerC and XerD in the presence and absence of PepA, at 10 and 40% glycerol. The DNA was nicked with DNase I or cleaved with XhoI, and products were separated on agarose gels (Figure 3C; data not shown). p-psiDC.psiDC recombined relatively efficiently in the presence of PepA (Figure 3C). This PepA-dependent recombination was more efficient at 40% glycerol than at 10% glycerol, and yielded four-noded catenanes as the major product. There was no detectable recombination in the absence of PepA at 10% glycerol, but at 40% glycerol there was a small amount of recombination, yielding a mixture of four-, six- and eight-noded catenanes. Restriction analysis confirmed that p-psiDC.psi gave the expected inversion product (data not shown). p-psiDC.psi recombined slightly less efficiently than p-psiDC.psiDC. In the presence of PepA, at 10 and 40% glycerol, the major product was three-noded knots, although traces of five- and seven-noded knots were detected at 40% glycerol. In the absence of PepA, low levels of recombination were observed at 40% glycerol; products were a mixture of three-, five- and seven-noded knots. This PepA-independent recombination on p-psiDC.psi was similar to the recombination observed between two psi cores in an inverted repeat on p-CD.DC (compare with Figure 2), supporting the hypothesis that there are two pathways of recombination: a PepA- and accessory sequence-independent pathway that is enhanced at 40% glycerol and gives products of mixed topology, and a more efficient pathway that requires PepA and two copies of the accessory sequences, giving products of a specific topology. The order of strand exchange is reversed at psiDC XerC cleaves and exchanges top strands of psi, adjacent to the XerC-binding site at the left end of the central region, whereas XerD cleaves and exchanges bottom strands at the right end of the central region (Figure 3A; Colloms et al., 1996; Blake et al., 1997). Xer recombination at psi proceeds with a defined order of strand exchanges (Colloms et al., 1996). XerC first exchanges top strands to generate a HJ intermediate. XerD then exchanges bottom strands to complete the reaction. If the order of strand exchange at psi is an intrinsic property of the way XerC and XerD bind to the psi core, then XerC will exchange the first pair of strands during recombination at both psi and psiDC. If, instead, the order of strand exchange at psi is determined solely by the architecture of the synapse formed by accessory proteins and sequences, then XerD will exchange the first pair of strands at psiDC. HJs made by XerC- and XerD-mediated strand exchange are not equivalent and can be distinguished by the recombinant strands they contain (Figure 4A). Strand-separating gel electrophoresis was used to reveal which strands had been exchanged in HJs formed during recombination between wild-type and inverted-core psi sites on p-psi.psi, p-psiDC.psi and p-psiDC.psiDC (Figure 4B). HJ yields were maximized by carrying out reactions at 40% glycerol, which leads to the accumulation of high levels of HJ intermediates in the presence of PepA (Hallet et al., 1999; this study). HJs were cleaved with XhoI to give χ-forms, and purified from an agarose gel. The χ-form HJs were 3′ end-labelled with 32P, and cleaved with restriction endonucleases that were predicted to give different sizes of single-stranded DNA fragments depending on which strands had been exchanged (Figure 4B). Samples were run on a denaturing agarose gel to analyse the separated single strands. Figure 4.Analysis of HJ intermediates formed by recombination at psi and psiDC. (A) HJ intermediates made by XerC or XerD strand exchange can be distinguished by the recombinant strands they contain. XhoI-cleaved χ-form HJs, 3′ end-labelled and then cleaved with restriction enzymes give different patterns of single-stranded DNA fragment depending on which strands have been exchanged. Accessory sequences are shown in blue, XerC- and XerD-binding sites are shown in green and pink, respectively, and 3′ end labels are indicated by asterisks. (B) XhoI-cleaved HJs from p-psi.psi, p-psiDC.psi and p-psiDC.psiDC were purified and 3′ end-labelled with 32P. Labelled χ DNA was run on a strand-separating agarose gel uncut or cleaved with HindIII, EcoRV or DraIII. Predicted sizes of labelled single-stranded DNA fragments for χ-forms produced by XerC or XerD strand exchange before or after cleavage with the appropriate restriction enzymes are tabulated. U indicates fragments unchanged by the restriction enzyme. Fragment sizes diagnostic for XerC or XerD strand exchanges are shown in bold. Sites taking part in each reaction are shown diagrammatically below each table. Curly arrows indicate recombinase partners initiating the first strand exchange reaction. Download figure Download PowerPoint The results of this analysis confirmed that HJs produced by recombination between two wild-type psi sites are formed by exchange of XerC strands, as previously reported (Figure 4B; Colloms et al., 1996). HJs produced by recombination between psi and psiDC were also formed mainly by exchange of XerC strands (Figure 4B). In contrast, recombination between two psiDC sites yielded HJs formed by exchange of XerD strands (Figure 4B). Thus the order of strand exchange at psi is determined by the orientation of the core with respect to the accessory sequences. During recombination at identical sites, the recombinase that binds closest to the accessory sequences (XerC for psi, XerD for psiDC) exchanges the first pair of strands. However, when the two cores are in opposite orientations with respect to the accessory sequences in p-psiDC.psi, it appears that the natural preference for XerC to initiate recombination at psi cores dictates the order of strand exchange. In addition to the major HJ with XerD strands exchanged in p-psiDC.psiDC, a small amount of HJ formed by XerC strand exchange was detected (Figure 4B). This probably comes from some PepA-independent recombination, which is known to occur in the high glycerol conditions used (see above). In this pathway, the location of the accessory sequences will have no effect and XerC strand exchange will probably occur first. Recombination at psiDC with catalytic mutants of XerC and XerD In the previous section, it was shown that XerD strands are exchanged in the HJ formed between two psiDC sites. However, it was not proven that XerD catalyses this strand exchange. It is possible that XerC binds to the XerD-binding site and exchanges XerD strands at psiDC. Catalytic mutants of XerC and XerD were therefore used to ascertain which recombinase carries out the first strand exchange at psiDC. In vitro recombination of p-psi.psi and p-psiDC.psi was completely abolished when wild-type XerC was replaced by the catalytically inactive mutant XerCY275F. In contrast, when XerD was replaced with XerDY279F, HJs formed by XerC-mediated strand exchange accumulated on both of these plasmids (data not shown; see also Colloms et al., 1996). However, no HJs or recombination products were detected on p-psiDC.psiDC with either XerCY275F and XerD, or XerC and XerDY279F. Similarly, no HJs or products were obtained from p-psiDC.psiDC with other recombinase catalytic mutants, such as XerCR148K or XerDR148K, in combination with wild-type partners. The failure to produce HJs from p-psiDC.psiDC with catalytic mutants of XerC might be due to reduced activity or DNA binding affinity of these XerC mutants, or altered interactions with XerD (Arciszewska et al., 2000). Therefore, to determine which recombinase exchanges the first pair of strands on p-psiDC.psiDC, recombination was examined in E.coli strains carrying chromosomal catalytic mutations in xerC or xerD (Table I). This approach is more sensitive because Xer recombination is more efficient in vivo than in vitro. HJs produced by Xer-mediated strand exchange in vivo can be processed, probably by replication or by HJ-resolving enzymes, to give fully recombinant product (Colloms et al., 1996). As expected, p-psi.psi recombined efficiently in the presence of XerC and XerDY279F, but not at all in the presence of XerCY275F and XerD. In contrast, p-psiDC.psiDC did not recombine in the presence of XerC and XerDY279F, but did recombine in the presence of XerCY275F and XerD. These results support the hypothesis that XerD exchanges the first pair of strands on p-psiDC.psiDC to give HJs that can be resolved in vivo to recombinant product. Table 1. In vivo recombination at psi and psiDC in E.coli strains carrying mutant xer genes Plasmid Genotype xerC+ xerD+ xerC+ xerDYF xerCYF xerD+ xerCYF xerDYF p-psi.psi +++ +++ − − p-psiDC.psiDC ++ − + − Plasmids were transformed into strains carrying the chromosomal copies of xer alleles indicated. The extent of recombination observed by agarose gel electrophoresis after overnight growth is indicated as: +++, 100% of substrate converted to product; ++, 50% of substrate converted to product; +, 15% of substrate converted to product; −, no detectable recombination. Topology of HJ intermediates The HJs produced in vitro at psiDC could be genuine intermediates in recombination, or they could be dead-end products from another pathway. If the HJs are genuine intermediates, their topology should be consistent with that of the fully recombinant products. HJs that are intermediates in the production of three-noded knots should have three trapped nodes and will co-migrate on agarose gels with a three-noded knot when singly nicked. Similarly, intermediates in the production of four-noded catenanes should have four entrapped catenation nodes and will co-migrate with a four-noded catenane (Colloms et al., 1996). The topology of HJs produced by XerC and XerD in the presence of PepA at 40% glycerol was determined by two-dimensional gel electrophoresis. Nicked products were separated according to their topology in the first dimension. Gel lanes were then excised and incubated with XhoI prior to electrophoresis in the second dimension, to ascertain the restriction patterns of the different products (Figure 5). This analysis showed that HJs produced in the reaction between psi and psiDC had three entrapped nodes, whereas HJs produced in the reaction between two psiDC sites had four entrapped nodes. Thus the HJs have the correct topology to be intermediates in the production of three-noded knots and four-noded catenanes, respectively. Figure 5.Two-dimensional gel analysis of HJs formed by recombination between psi and psiDC sites. (A) Analysis of products formed by recombination on p-psiDC.psi. (B) Analysis of products formed by recombination on p-psiDC.psiDC. The first dimension separated substrates and products according to their topology, and the second dimension separated products according to their restriction pattern. Arrows indicate the positions of HJ intermediates on each gel. Migration of three-noded knots, four-noded catenanes and other species in the first dimension is indicated to the left of a duplicate first- dimension lane as in the legend to Figure 2. Second-dimension mobilities are shown above the gels as HJ, HJ intermediates; L, linear substrate; S, substrate restriction fragment; and P, product restriction fragment. Small product and substrate restriction fragments are not shown. Partial digestion with XhoI gave some linear substrate at the oc S position, and some uncut (open circle) large product circle at the four-node position in (B). Download figure Download PowerPoint Recombination at psiDC with XerC[De] A derivative of XerC (XerC[De]), in which the C-terminal 21 residues of XerC have been replaced with the C-terminal 17 residues from XerD, has been shown to stimulate XerD strand exchange and impair XerC strand exchange, when tested on synthetic HJs (H.Ferreira and L.K.Arciszewska, unpublished results). Replacing XerC with XerC[De] inhibited in vitro recombination on p-psi.psi and p-psiDC.psi, but strongly stimulated recombination on p-psiDC.psiDC, giving increased yields of both HJ and recombinant product in a standard 1.5 h reaction (Figure 6A; data not shown). This result is consistent with the 'stimulation of partner, impairment of self-catalysis' phenotype of XerC[De]. Stimulation of XerD and impairment of XerC function gave higher amounts of HJs on p-psiDC.psiDC where XerD strand exchange occurs first. In contrast, the reduced activity of XerC[De] gave lower yields of HJs, and consequently lower levels of final recombination product on p-psi.psi and p-psiDC.psi where XerC strand exchange is first. Figure 6.Xer recombination at psi and psiDC in the presence of XerC[De]. (A) Recombination of p-psi.psi and p-psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. (B) Time course of recombination on p-psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with XhoI. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments. Download figure Download PowerPoint A time course of recombination on p-psiDC.psiDC using XerC[De] and wild-type XerD showed that HJs formed by XerC[De] and XerD are transient recombination intermediates which become resolved to the final product. Large amounts of HJs are observed at early time points, and gradually disappear at later time points (Figure 6B). HJs and products formed in this reaction have the same four-noded topology as those produced by wild-type XerC and XerD on this substrate (data not shown). Strand-separating gels confirmed that the HJs formed by XerC[De] and XerD on p-psiDC.psiDC were formed by XerD strand exchange (data not shown). These experiments therefore confirm that HJs in which XerD strands have been exchanged are intermediates in a complete recombination reaction between two psiDC sites. Discussion The results presented here demonstrate that two sets of accessory sequences are required for topological selectivity and efficient recombination at psi. The psi core appears to have been selected to be almost completely inactive in the absence of accessory sequences and accessory proteins. Recombination occurs only after the formation of the productive synapse, which acts as a checkpoint to ensure that sites are in a direct repeat in the same DNA molecule. This reflects the biological function of psi, which is to resolve plasmid multimers. A very small amount of accessory factor-independent recombination was observed at psi cores, consistent with the low level of recombination between psi cores previously seen in vivo (Blake et al., 1997). This recombination did not give products of a specific topology, showing that two full sets of accessory sequences are necessary for the formation of an interwrapped synapse. In contrast, Tn3 resolvase uses a similar mechanism to ensure that sites are only recombined if they are in a direct repeat on the same DNA molecule, but one set of res accessory sequences was enough to restrict the product topology to a single species (Bednarz et al., 1990). Recombination between two psiDC sites occurred with the same topology as recombinatio