Oct 2, 1992 - Lloyd and Buckman, 1991; Kalman et al., 1992). The mutant phenotype is very modest and is masked by the activities of the ruv genes. Thus ...
The EMBO Journal vol. 12 no. 1 pp. 17 - 22, 1993
Dissociation of synthetic Holliday junctions by E.coli RecG protein
Robert G.Lloyd1 and Gary J.Sharples Department of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK ICorresponding author Communicated by D.M.J.Lilley
The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation. The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes. RuvA and RuvB act together to promote branch migration of Holiday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage. In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry. We show that the 76 kDa RecG protein is a DNA-dependent ATPase, like RuvB. Using gel retardation assays we demonstrate that it binds specifi'cally to a synthetic Holliday junction, like RuvA and RuvC. Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB. We suggest that RecG and RuvAB provide alternative activities that can promote branch migration of Holliday junctions in recombination and DNA repair. Key words: ATPase/branch migration/Holliday junctions/ recombination/repair
Introduction The recG locus is required for normal recombination and DNA repair in Escherichia coli. Strains carrying mutations in this gene show reduced recombination in conjugational crosses and increased sensitivity to ultraviolet light, mitomycin C and ionizing radiation (Storm et al., 1971; Lloyd and Buckman, 1991; Kalman et al., 1992). The mutant phenotype is very modest and is masked by the activities of the ruv genes. Thus, while a recG or ruv mutation reduces conjugational recombination by no more than -3-fold, a recG ruv double mutation reduces recombination by > 500-fold. The double mutants are also extremely sensitive to ultraviolet light, much more so than the single mutants (Lloyd, 1991). This synergism suggests a possible functional overlap between the products of recG and ruv. Three genes have been identified at the ruv locus. Mutations in these genes confer very similar deficiencies in DNA repair and recombination (Otsuji et al., 1974; Lloyd et al., 1984; Iwasaki et al., 1989b; Sargentini and Smith, 1989; Sharples et al., 1990). ruvA and ruvB form an operon and Oxford University Press
are regulated by LexA repressor as part of the SOS response to DNA damage (Shurvinton and Lloyd, 1982; Benson et al., 1988; Shinagawa et al., 1988). ruvC is located just upstream of ruvAB in a separate operon with another gene (orf-26) of unknown function (Sharples et al., 1990; Sharples and Lloyd, 1991; Takahagi et al., 1991). The products of all three ruv genes have been overproduced and purified. RuvA binds to DNA and forms specific complexes with synthetic Holliday junctions that serve as models for intermediates in recombination (Shiba et al., 1991; Parsons et al., 1992; Tsaneva et al., 1992b). RuvB is a DNAdependent ATPase whose activity is enhanced by RuvA (Iwasaki et al., 1989a; Shiba et al., 1991). These two proteins also provide an activity that can dissociate Holliday junctions by catalysing branch migration (Shiba et al., 1991; Parsons et al., 1992; Tsaneva et al., 1992a). RuvC is an endonuclease that binds specifically to Holliday junctions and cleaves these junctions to produce recombinant products (Connolly et al., 1991; Dunderdale et al., 1991; Iwasaki et al., 1991). The properties of the Ruv proteins are consistent with earlier genetic studies, which suggested that ruv defines activities needed at a late stage in recombination to convert intermediates into viable products (Lloyd et al., 1984; Clark and Low, 1988; Benson et al., 1991). The overlap between recG and ruv led us to consider the possibility that RecG protein may also be involved in processing recombination intermediates. We have purified RecG and shown that it is a DNA-dependent ATPase that binds a synthetic Holliday junction and dissociates the structure. The reaction products suggest that RecG has a branch migration activity similar to that catalysed by RuvAB. We propose that RecG and RuvAB provide reasonably efficient alternatives for recombination in genetic crosses and also that RuvAB takes over the major role in repair following SOS induction of these proteins.
Results Overproduction and purification of RecG The recombinant plasmid pGS772 carries the coding region for recG downstream of the 410 promoter of pT7-7. The 76 kDa RecG protein was overexpressed from this plasmid in strain GS1269 by incubation with isopropyl ,B-Dthiogalactoside (IPTG) to induce T7 RNA polymerase (Figure 1, lane c). Purification of RecG from the induced cells was followed by SDS -PAGE. The cell lysate was centrifuged and the supernatant was passed through a DEAEBio-Gel A column (>90% of RecG appeared in the flow through, Figure 1, lane d) and purified by chromatography on single-stranded DNA (ssDNA) cellulose and hydroxylapatite columns. Purification was simplified by the fact that RecG was the major protein to bind to ssDNA cellulose. The only visible contaminant in the pooled RecG fractions from this column was a low molecular weight species, which 17
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98% RecG by reversed phase HPLC. The sequence of the amino-terminus was analysed using a pulsed liquid amino acid sequencer (model 473, Applied Biosystems). The first nine residues from the amino terminus matched the MetLys-Gly-Arg-Leu-Leu-Asp-Ala-Val predicted by the DNA sequence of recG (Lloyd and Sharples, 1991).
RecG is a DNA-dependent ATPase The sequence predicted for RecG contains a motif that is well conserved in proteins that bind ATP (Walker et al., 1982; Gorbalenya et al., 1988; Lloyd and Sharples, 1991). The purified protein was found to be a potent ATPase in the presence of single-stranded DNA, supercoiled DNA, or linear duplex DNA (Figure 2; data not shown). The turnover number for ATP hydrolysis was calculated to be 3500/min with supercoiled DNA as a cofactor. By comparison, very little activity was seen in the absence of DNA. ATPase activity was also monitored during the purification of RecG. The peak of ATPase coincided with the peak of RecG during chromatography on hydroxylapatite (data not shown). -
RecG binds to a synthetic Holliday junction Since there is a functional overlap between recG and the ruv genes (Lloyd, 1991), we examined the ability of RecG to bind to a Holliday junction. We used the synthetic Xjunction, which has been described previously for studies of the Ruv proteins (Dunderdale et al., 1991; Parsons et al., 18
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Fig. 3. Gel retardation of a synthetic Holliday junction. Increasing concentrations of purified RecG were mixed on ice with 0.15 yM 5,-32P-labelled junction or linear duplex DNA. Complexes were separated on a low ionic strength gel at room temperature and labelled DNA was detected by autoradiography.
1992). When the junction was incubated with increasing amounts of RecG and electrophoresed on a low ionic strength polyacrylamide gel, a well defined protein-DNA complex was observed (Figure 3, lanes b-h). No binding to linear duplex DNA was observed under these assay conditions (lanes j-1). RecG was able to form a protein-DNA complex with the junction at concentrations of monomeric protein that were significantly lower than those needed under identical conditions with either RuvA or RuvC (Figure 4). RuvA forms two complexes, as reported previously (Parsons et al., 1992). The single RecG complex migrated faster than the RuvA complexes, but slower than the RuvC complex. A faint smear migrating ahead of the RecG complex suggests that it is somewhat less stable than the complexes formed by RuvA and RuvC. RecG dissociates a synthetic junction We next considered the possibility that RecG might be able to dissociate a synthetic Holliday junction in a manner similar to that reported for RuvAB (Parsons et al., 1992). To test this possibility, RecG was incubated with the synthetic Holliday junction at 37°C in the presence of ATP and the
E.coli RecG
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products clectrophoresed on a 10% native polyacrylamide gel. With increasing concentrations of protein, junction DNA disappeared and was replaced by two new DNA bands migrating very close together (Figure 5, lanes b-e). The mobility of these bands was intermediate between that of the junction and linear duplex DNA, and coincided exactly with that of two 'split-end' duplex markers made by annealing 32P-labelled oligonucleotide 1 with RuvA oligonucleotides 2 and 4, respectively. The top band migrated with marker 1 + 4 (lane 1), while the bottom band migrated opwq- with marker 1 + 2 (lane k). These markers are identical to the products expected from dissociation of the synthetic Holliday junction by branch migration (Figure 6). The additional very faint band migrating just ahead of the junction in the RecG reactions (Figure 5, lanes b-e) was identified as the product of annealing oligonucleotide 4 to reaction product 1 + 2 (lane n; data not shown). Under the conditions of the assay, product 1 + 4 was a little unstable and released RuvC oligonucleotide 4 during the reaction (Figure 5, lane 1). The 'split-end' duplex markers were not altered by incubation with RecG under the reaction conditions, with or without ATP (data not shown). Dissociation of the junction shows an absolute requirement for ATP and Mg2 + (Figure 5, lanes f and g). No dissociation was observed when ATP was replaced by ATP-y-S (data not shown), which implies that ATP hydrolysis is necessary. The reaction with ATP is very efficient. Dissociation of the junction under the standard assay conditions is detectable within 15 min even when the RecG C ~:3 ~Hconcentration of RecG is reduced to as little as 20 pM (data not shown). At low protein levels (0.02-2.0 nM RecG) the reaction seemed to favour the production of 'split-end' duplex 1 + 4 (data not shown). The dissociation of the junction was further examined by sampling the reaction at different Fig. 4. Effect of protein concentration orn the formation of times. Since RecG is a strong ATPase, the time-course was protein-Holliday junction complexes by RuvA, RuvC and RecG. The conducted with and without ATP regeneration. From the data formation of complexes with the 5,_32p_lF abelled junction was assayed obtained (Figure 7), it is clear that 'split-end' duplex 1 + as in Figure 3. 4 is the favoured product at early times. ATP regeneration appears to have little effect. - .*.^ -The dissociation of a synthetic Holliday junction by RecG .. is very similar to the reaction catalysed by RuvAB (Parsons r.l et al., 1992). The only significant difference is that %J, dissociation by RuvAB requires high levels of protein, E: --': A 5, r, 1 particularly of RuvB. Given the genetic interaction between recG and all three ruv genes, we next asked whether RecG, like RuvC, is also able to resolve a synthetic junction by endonuclease cleavage. RuvC cleaves a synthetic Holliday junction to produce nicked duplex products (Connolly et al., 1991; Dunderdale et al., 1991). We have not seen this type toso0 ~ " -- < of product in reactions with RecG, with or without ATP (Figures 5 and 7; data not shown). RuvC resolves a synthetic Holliday junction rather poorly (Dunderdale et al., 1991; Iwasaki et al., 1991). The addition of RecG to the RuvC reaction does not stimulate cleavage and when ATP is also present, no cleavage is detected and the junction is instead dissociated into the two 'split-end' duplex products seen with Fig. 5. Dissociation of synthetic Holliday junctions by RecG. RecG alone (data not shown). At high concentrations of Reactions containing 0.15 1tM 5'-32P-labe lied junction or linear duplex RecG (> 100 nM), we detected some exonuclease activity DNA were incubated for 60 min at 37°C with RecG, ATP, MgC12 (data not shown). We suspect that this activity is due to a and EDTA, present or absent as indicatedJ (lanes a-j). Marker DNAs minor contaminant in our RecG preparation since it could (lanes k-m) were incubated in parallel in reaction buffer. Lane n be removed entirely by gel filtration (data not shown). The contains a mixture of the two markers ma oligonucleotides + 2 and + 4. React ions were stopped and nuclease-free RecG was examined and found to have the deproteinized as described in Materials anid methods, and the products same ATPase, junction binding and junction dissociation analysed on a 10% polyacrylamide gel. activities as the original preparation (data not shown).
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Fig. 6. Diagram showing the products expected from branch migration of a synthetic Holliday junction. A. Structure of the junction used showing the 12 bp homologous core (solid lines) and the 18-20 bp heterologous arms (shaded lines). The 32P-labelled 5' end of oligonucleotide 1 is indicated by an asterisk. B. Binding of RecG and branch migration to the left producing labelled product 1 + 2 and to the right producing labelled product 1 + 4.
Discussion We have overproduced and purified RecG and shown that it is a DNA-dependent ATPase that binds to a synthetic Holliday junction and dissociates the Holliday structure to duplex products that are characteristic of a branch migration reaction. These properties are shared with the RuvAB proteins, though there are several notable differences. First, and most obviously, the activities reside within a single polypeptide of 76 kDa. In the case of RuvAB, the ability to bind specifically to a Holliday junction is specified by the 22 kDa RuvA subunit. The ATPase activity is provided by the 37 kDa RuvB subunit, which probably drives branch migration (Iwasaki et al., 1989a; Shiba et al., 1991; Parsons et al., 1992). RuvB can dissociate junctions on its own, but this requires large amounts of protein (Tsaneva et al., 1992b). Presumably, RuvA normally serves to target RuvB to the junction (Parsons et al., 1992). RecG is also a much more potent ATPase than RuvB, especially in the presence of DNA. The RuvB activity is stimulated by DNA, but only if RuvA is also present (Shiba et al., 1991). The very potent ATPase of RecG, coupled with the fact that it dissociates a synthetic Holliday with heterologous arms, is indicative of a DNA helicase activity. The protein sequence contains several motifs that are shared with a number of DNA and RNA helicases (Lloyd and Sharples, 1991). However, we failed to see such an activity using a conventional helicase substrate made by annealing an oligonucleotide to circular single-stranded DNA (Matson and Kaiser-Rogers, 1990). The results presented here show that RecG is also unable to unwind linear duplex molecules with unpaired strands at one end. It is possible that the activity of RecG is targeted to specialized structures such as Holliday junctions, as has been argued for RuvAB (Parsons et al., 1992). With low protein concentrations, dissociation of junctions by RecG was
20
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Fig. 7. Dissociation of synthetic Holliday junctions: time-course and effect of ATP regeneration. Reaction mixtures with (lanes a-f) and without (lanes g-1) an ATP regeneration system were scaled up to 150 11. At the times indicated, 20 pl samples were removed, deproteinized and the products analysed on an 8% polyacrylamide gel as described.
favoured in one of the two possible directions. We do not know if this bias is a feature of RecG or is a consequence of the DNA sequence of the junction used. The possibility that both RecG and RuvAB can catalyse branch migration of Holliday junctions would provide a simple explanation for the functional overlap between recG and ruvAB seen previously in vivo (Lloyd, 1991). The available evidence suggests that both activities can catalyse recombination fairly efficiently. Both recG and ruvAB strains are reasonably proficient in recombination (Lloyd et al., 1984, 1987; Lloyd, 1991; Lloyd and Buckman, 1991). However, there is a slight deficiency in recombination in both cases, which indicates that the two activities are not
E.coli RecG protein
fully interchangeable and compensate rather imperfectly for one another. The situation with repair may be different. ruvAB mutants are far more sensitive to UV light than recG mutants (Lloyd, 1991; Lloyd and Buckman, 1991). RuvAB may therefore play the more important role during recombinational repair of DNA strand gaps (West et al., 1981). The increased synthesis of RuvAB following SOS induction would be consistent with this view (Shurvinton and Lloyd, 1982). We failed to detect any cutting of a synthetic Holliday junction by RecG under conditions where the structure is resolved by RuvC into nicked duplex products. When both proteins were present in the same reaction and ATP was provided, no cleavage was detected and the junction was dissociated by branch migration as in reactions with RecG alone. This observation is consistent with the fact that RecG appears to have a higher affinity for junction DNA and dissociates the junction very rapidly. By contrast, the specific activity of RuvC nuclease is known to be rather modest (Dunderdale et al., 1991; Iwasaki et al., 1991). However, from the data presented we cannot rule out the possibility that RecG cleaves junctions under other conditions. It should be emphasized that functional overlap between recG and ruv extends equally to both ruvAB and ruvC (Lloyd, 1991). It is possible that some other as yet unidentified nuclease acts with RecG to provide a resolvase that can act as an alternative to RuvC. Alternatively, RecG may be activated in vivo to function as a nuclease.
Materials and methods Strains and plasmids Ecoli strain GS1269 is BL21(DE3) plysS (Studier and Moffat, 1986) transformed with the recG+ plasmid, pGS772. N2731 is a recG258 derivative of AB 1157 (Lloyd and Buckman, 1991). pBL 136 is a recG+ recombinant of pTZ19R (Lloyd and Sharples, 1991). DNA Synthetic Holliday junction DNA was made by annealing oligonucleotides I (5'-GACGCTGCCGAATTCTGGCTTGCTAGGACATCTTTGCCCACGTTGACCC-3'), 2 (5'-TGGGTCAACGTGGGCAAAGATGTCCTAGCAATGTAATCGTCTATGACGTT-3'), 3 (5'-CAACGTCATAG-
ACGATTACATTGCTAGGACATGCTGTCTAGAGACTATCGA-3'), and 4 (5'-ATCGATAGTCTCTAGACAGCATGTCCTAGCAAGCCAGAATTCGGCAGCGT-3') as described by Parsons et al. (1990). Linear duplex DNA was made by annealing oligonucleotides 1 and 5 (5'-GGGT-
CAACGTGGGCAAAGATGTCCTAGCAAGCCAGAATTCGGCAGCGTC-3'). Partially duplex markers with unpaired strands at one end ('split-end' duplexes 1 + 2 and 1 + 4) were prepared by annealing oligonucleotides 1 plus 2 and 1 plus 4. In all cases, oligonucleotide 1 was 32P-end-labelled at the 5' end before annealing. Circular FX174 viral (+) strand and supercoiled RF DNAs were purchased from Pharmacia LKB. DNA concentrations are in nucleotide equivalents. Junction DNA was measured using DNA DipSticks (Invitrogen, San Diego) and is approximate due to the low concentration.
Construction of a plasmid that overproduces RecG The recG gene was amplified from pBL136 with AmpliTaq polymerase (Perkin Elmer Cetus) using a standard PCR protocol and the oligonucleotide primers 5'-GGTAAGTCATATGAAAGGTC-3' and 5'-CGGCAGGAAGC77GGGTAAC-3' to incorporate NdeI and HindIlI sites (italicized) at the 5' and 3' ends, respectively. The PCR product was gel purified, cleaved with NdeI and HindlIl and ligated into pT7-7, a pBR322 based vector designed for expression of cloned genes from a phage T7 promoter (Tabor and Richardson, 1985). The ligation mixture was transformed into strain N2731 (recG) and ampicillin-resistant transformants carrying the appropriate recG+ construction were identified by their resistance to mitomycin C and restriction analysis of the plasmid DNA. The recG+ construct, pGS772, was introduced into strain BL21(DE3) plvsS. The resulting strain (GS 1269)
was shown by SDS -PAGE to overproduce a 76 kDa protein in response to IPTG, which we assumed to be RecG.
Purification of RecG Strain GS1269 was grown at 37°C to an OD650 of 0.5 in 5 1 of LB medium containing chloramphenicol and ampicillin. IPTG was added to a final concentration of 2 mM and incubation was continued for 3 h before harvesting the cells by centrifugation. The cell paste was washed in lysis buffer (100 mM Tris-HCI, pH 8.0, 2 mM EDTA and 5% glycerol) and resuspended in a final volume of 65 ml before storing at -80°C. The cells were thawed slowly at 4°C and incubated with lysozyme (0.2 mg/ml) at 37°C for 15 min in the presence of 0. IM NaCl, 10 mM (3-mercaptoethanol and 10 tzg/ml DNase I, before adding Triton X100 to 1% and PMSF to 1 mM. Incubation was continued at room temperature for 15 min before chilling the lysate on ice. All subsequent procedures were at 4°C. The lysate was centrifuged at 40 000 r.p.m. for 60 min in a Kontron TST 41 rotor. The supernatant fraction (56 ml) was dialysed for 3.5 h against buffer A [20 mM Tris-HCI, pH 8.5, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 10% glycerol] containing 0.5 mM PMSF. Soluble proteins were loaded onto a 65 ml DEAE-Bio-Gel A (Bio-Rad) column equilibrated with the same buffer. The flow through (2.8 mg/ml protein), which contained >90% of the RecG, was loaded directly onto a 25 ml ssDNA cellulose (Sigma) column equilibrated in buffer A, pH 7.5, and bound proteins were eluted with a 250 ml linear gradient of KCI in the same buffer at a flow rate of 1 ml/min. Fractions containing RecG, which eluted between 260-460 mM KCI (as identified by SDS-PAGE), were pooled (60 ml, 0.21 mg/ml protein), dialysed against buffer P (10 mM potassium phosphate, pH 6.8, 0.5 mM DTT, 150 mM KCI, 10% glycerol) for 3.5 h and loaded onto a 20 ml hydroxylapatite (Bio-Rad) column equilibrated in the same buffer. Bound proteins were eluted with a 240 ml linear gradient of phosphate buffer (10-600 mM potassium phosphate, pH 6.8, 0.5 mM DTT, 150 mM KCI and 10% glycerol) at a flow rate of 1 ml/min. Fractions containing RecG alone, which eluted between 20-140 mM phosphate (as identified by SDS-PAGE), were pooled and dialysed against buffer A, pH 7.5, 50% glycerol for 5 h and stored in aliquots at -80°C (17 ml, 240 Ag/ml). RuvA and RuvC proteins E. coli RuvA and RuvC proteins were purified as described by Dunderdale et al. (1991) and Tsaneva et al. (1992b). A TPase assay The release of [32P]ADP from [ca-32P]ATP was measured by thin layer chromatography on PEI-cellulose as described by Kornberg et al. (1978). Reaction mixtures (20 Al) contained 50 mM Tris-HCI, pH 7.5, 5 mM MgCl2, 1 mM DTT, 1 mM ATP, 0.01-0.05 yCi [a-32P]ATP (30 Ci/mmol, Amersham), DNA as required and 1-5 Al protein sample. Reactions were incubated for 30 min at 37°C. For time-courses, reactions were set up in bulk and 20 yl samples removed at the times indicated. Reactions were stopped by quenching with 5 pl 0.5 M EDTA.
Gel retardation assay Reaction mixtures (20 pA) contained 32P-labelled synthetic Holliday junction or linear duplex DNA (-0.15 AM) in binding buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM DTT and 100 Ag/ml bovine serum albumin) and various amounts of the test protein. After 15 min on ice, 5 pA loading buffer (40 mM Tris-HCI, pH 7.5, 4 mM EDTA, 25% glycerol and 400 pg/ml bovine serum albumin) were added and the samples loaded immediately onto 4% polyacrylamide gels in low ionic strength buffer (6.7 mM Tris-HCI, pH 8.0, 3.3 mM sodium acetate and 2 mM EDTA). Electrophoresis was carried out at room temperature for 1.75 h at 200 V with continuous circulation of buffer. Gels were dried on Whatman 3MM paper and autoradiographed. Dissociation of a synthetic Holliday junction Reaction mixtures (20 p1I) contained synthetic Holliday junction or linear duplex DNA ( - 0.15 AM) in reaction buffer (50 mM Tris-HCI, pH 8.0, 1 mM DTT, 5 mM MgCl,, 100 Agg/ml bovine serum albumin and ATP as specified) and various amounts of protein sample. An ATP regeneration system was provided where indicated by including 20 mM phosphocreatine and 6 units/ml phosphocreatine kinase in the reaction. Reactions were incubated at 37°C for the indicated times before adding 5 Al stop buffer [2.5% (w/v) SDS, 200 mM EDTA and 10 mg/ml proteinase K] and incubating for a further 10 min at 37°C. The DNA products were then electrophoresed at room temperature on an 8 or 10% native polyacrylamide gel using a Tris - borate buffer system (Parsons et al., 1990) followed by
autoradiography.
21
R.G.Lloyd and G.J.Sharples
Acknowledgements We would like to thank John Keyte for sequencing the amino-terminus of RecG and for synthesizing the oligonucleotides used and Lynda Harris for technical assistance. We are particularly grateful to Matthew Whitby for help and encouragement and also to Stephen West and members of his laboratory at the Imperial Cancer Research Fund, Clare Hall for advice and useful hints.
References Benson,F.E., Illing,G.T., Sharples,G.J. and Lloyd,R.G. (1988) Nucleic Acids Res., 16, 1541-1549. Benson,F., Collier,S. and Lloyd,R.G. (1991) Mol. Gen. Genet., 225, 266-272. Clark,A.J. and Low,K.B. (1988) In Low,K.B. (ed.) The Recombination of Genetic Material. Academic Press, New York, pp. 155 -215. Connolly,B., Parsons,C., Benson,F.E., Dunderdale,H.J., Sharples,G.J., Lloyd,R.G. and West,S.C. (1991) Proc. Natl. Acad. Sci. USA, 88, 6063-6067. Dunderdale,H.J., Benson,F.E., Parsons,C.A., Sharples,G.J., Lloyd,R.G. and West,S.C. (1991) Nature, 354, 506-510. Gorbalenya,A.E., Koonin,E.V., Donchenko,A.P. and Blinov,V.M. (1988) Nature, 333, 22-23. Iwasaki,H., Shiba,T., Makino,K., Nakata,A. and Shinagawa,H. (1989a) J. Bacteriol., 171, 5276-5280. Iwasaki,H., Shiba,T., Nakata,A. and Shinagawa,H. (1989b) Mol. Gen. Genet., 219, 328-331. Iwasaki,H., Takahagi,M., Shiba,T., Nakata,A. and Shinagawa,H. (1991) EMBO J., 10, 4381-4389. Kalman,M., Murphy,H. and Cashel,M. (1992) Gene, 110, 95-99. Komberg,A., Scott,J.F. and Bertsch,L.L. (1978) J. Biol. Chem., 253, 3298-3304. Lloyd, R.G. (1991) J. Bacteriol., 173, 5414-5418. Lloyd,R.G. and Buckman,C. (1991) J. Bacteriol., 173, 1004-1011. Lloyd,R.G. and Sharples,G.J. (1991) J. Bacteriol., 173, 6837-6843. Lloyd,R.G., Benson,F.E. and Shurvinton,C.E. (1984) Mol. Gen. Genet., 194, 303-309. Lloyd,R.G., Buckman,C. and Benson,F.E. (1987) J. Gen. Microbiol., 133, 2531 -2538. Matson,S.W. and Kaiser-Rogers,K.A. (1990) Annu. Rev. Biochem., 59, 289-329. Otsuji,N., Iyehara,H. and Hideshima,Y. (1974) J. Bacteriol., 117, 337-344. Parsons,C.A., Kemper,B. and West,S.C. (1990) J. Biol. Chem., 265, 9285-9289. Parsons,C.A., Tsaneva,I., Lloyd,R.G. and West,S.C. (1992) Proc. Natl. Acad. Sci. USA, 89, 5452-5456. Sargentini,N.J. and Smith,K.C. (1989) Mutat. Res., 215, 115-129. Sharples,G.J. and Lloyd,R.G. (1991) J. Bacteriol., 173, 7711-7715. Sharples,G.J., Benson,F.E., Illing,G.T. and Lloyd,R.G. (1990) Mol. Gen. Genet., 221, 219-226. Shiba,T., Iwasaki,H., Nakata,A. and Shinagawa,H. (1991) Proc. Natl. Acad. Sci. USA, 88, 8445-8449. Shinagawa,H., Makino,K., Amemura,M., Kimura,S., Iwasaki,H. and Nakata,A. (1988) J. Bacteriol., 170, 4322-4329. Shurvinton,C.E. and Lloyd,R.G. (1982) Mol. Gen. Genet., 185, 352-355. Storm,P.K., Hoekstra,W.P.M., De Haan,P.G. and Verhoef,C. (1971) Mutat. Res., 13, 9-17. Studier,F.W. and Moffat,B. (1986) J. Mol. Biol., 189, 113-130. Tabor,S. and Richardson,C.C. (1985) Proc. Natl. Acad. Sci. USA, 82, 1074-1078. Takahagi,M., Iwasaki,H., Nakata,A. and Shinagawa,H. (1991) J. Bacteriol., 173, 5747-5753. Tsaneva,I.R., Muller,B. and West,S.C. (1992a) Cell, 69, 1171-1180. Tsaneva,I., Mlling,G., Lloyd,R.G. and West,S.C. (1992b) Mol. Gen. Genet., in press. Walker,J.E., Saraste,M., Runswick,M.J. and Gay,N.J. (1982) EMBO J., 1, 945-951. West,S.C., Cassuto,E. and Howard-Flanders,P. (1981) Nature, 294, 659-662.
Received on Septernber 2, 1992; revised on October 2, 1992
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