Genetical and biochemical evidence for the involvement of the coprotease domain of Escherichia coli RecA protein in recombination

J. Mol. Biol. (1992) 223, 823-829

Genetical and Biochemical Evidence for the Involvement of the Coprotease Domain of Escherichia coli RecA Protein in Recombination Christophe Cazaux and Martine Defais Laboratoire de Pharmacologic et de Toxicobgie Fondamentales C.N.R.S., 205 route de Narbonne, 31077 Toulouse Cedex, France (Received 15 May

1991; accepted 4 November 1991)

RecA amino acid residue 204 is involved in the coprotease domain of the protein responsible for the induction of mutagenic repair. Two mutations were created at this site leading to the addition of either a methyl or an isopropyl group on the original glycine. Analyses of bath the in vivo and the in vitro properties of these mutated proteins demonstrated that this residue 204 is involved in many RecA activities, suggesting that this site could allosterically direct conformational changes in the protein or could be situated in a region interacting with many RecA cofactors. Keywords: RecA; recombination;

SOS Repair; strand-exchange

Escherichia coli RecA protein carries out two main functions: it is required for homologous recombination and post-replication repair (Clark, 1973), and it acts as a protease cofactor facilitating LexA-repressor cleavage, which leads to the induction of the SOS repair response when E. coli is exposed to DNA-damaging agents (Walker, 1984). RecA protein is involved in mutagenesis not only by controlling the posttranscriptional proteolytic activation of UmuD protein (Burckardt et al., 1988; Shinagawa et al., 1988) but also by exerting another as yet unknown role (Nohmi et al., 1988; Dutreix et al., 1989; Sweasy et aZ., 1990). In addition RecA protein participates in a few SOS functions, such as stable replication (Witkin & Kogoma, 1984) and induced replisome recovery (Khidhir et al., 1985). Thus, RecA protein performs multiple roles in E. coEi (Walker, 1984; Cox & Lehman, 1987). which suggests the presence of several functional domains in the protein. Many mutants of RecA protein have been characterized (Clark & Margulies, 1965; Blanc0 et al., 1975) and recently new mutants have been isolated, some of them identifying separate potential domains for coprotease and recombinase activities (Kawashima et al., 1984; Yarranton & Sedgwick, 1982; Larminat & Defais, 1989; Dutreix et aE., 1989; Cazaux et al., 1991) and others indicating that both domains may overlap in the vicinity of amino acid residue 204 (Wang & Tessman, 1986). Among the former, the recA430 mutation, where glycine residue 204 is replaced by a serine residue (Kawashima et aZ., 1984), is of particular interest since it reduces protease activity, as OO22-2836/92/0400823-07

$03.OWO

reaction; mutagenesis

shown by a decreased induction of both SOS response and prophage (Blanc0 et al., 1975; Devoret et al., 1983), and an inefficient UmuD cleavage (Nohmi et al., 1988) while it preserves recombination activity (Morand et al., 1977). The molecular bases of these effects have been investigated in vitro. It was first suggested that the primary defect of RecA430 protein was an impaired formation of the complex between the protein and single-stranded (sst) DNA in the presence of ATP (Wabiko et al., 1983). Then Ikawa et al. (1989) demonstrated that the binding itself of RecA430 protein to ssDNA was normal, but that the defect came from an inability of the protein to form a ternary complex by homology-independent conjunction of single and doublestranded (ds) DNA molecules. Recently it has been demonstrated that the steady-state affinity and the rate of association of RecA430 protein to ssDNA was reduced (Menetski & Kowalczykowski, 1990). ln order to understand better the functional role of the RecA region around residue 204 in both SOS repair and recombination, minor changes have been created three and seven residues downstream from GIy204 (Cazaux et al., 1991). These mutations strongly affect. all the in vivo and in vitro activities of RecA protein. In addition, the mutation recA142, which is mostly affected in recombination, has been located at residue 225 (Dutreix et al., 1985, 1989;

t Abbreviations used: ss, single-stranded; ds. doublestranded; u.v.. ultra-violet light; RecAwt, wild type RecA protein: SSB, single-stranded-DXA4 binding. 0

1992 Academic

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Table 1 In vivo

Allele

phenotypes

Survivalt (%)

of recA

mutant

SOS induction1

wtreeA recA430

100 0.7

50 1.2

AWA

< 0.0 1

I.0

wcA604 recA605

1.1 0.3

2.5 12

alleles Intrachromosomal recombination$ (mating cross-efficiency) 100 (100) 27 (100) Null

24 (92) 03 (6)

The recA430 gene is carried by plasmid pREU46 derived from pBEU2 (Uhlin et al., 1983) and prepared from E. coli strain JC10289 (kindly provided by A. J. Clark). Mutations recA604 and recA605 were constructed as previously described (Cazaux et al., 1991). The Ml3 phage DNA BamHI fragments carrying the recA604, recA605 or wild-type gene were cloned into the unique BarnHI site in pRR322 (Bolivar et al., 1977) to produce, respectively, pCC604. pCC605 and pFL352 plasmids. Plasmid-harbouring cells were grown with 100 pg ampicillin/ml in Luria broth (Difco) except for the SOS induction, for which M63 minimal medium, supplemented with 0,4e/, (w/v) glucose, 65’j/, (w/v) casamino acid and 1 pg thiamine/ml, was used (Miller, 1972). t u.v.-irradiation (30 J/m’) and cell survival assays were previously described (Calsou t Defais, 1985). Strain used was ,JC10289 that had been deleted for the chromosomal recA gene (Csonka et al., 1979). $ SOS induction was determined as previously described (Larminat & Defais, 1989). The induction factor is the ratio of /?-galactosidase activities after and before irradiation (Miller, 1972). Cells were exposed to 10 J/m2 and incubated at 37°C for 90 min after which tea : : la& induction was measured. 5 For the genetic recombination assay, 2 different methods were used. Intrachomosomal recombination was measured as described (Dutreix et al., 1989) using stain FL7023, which is derived from GY7023 and carries the deletion A(recA-srlR)306. Conjugational recombination (values in parentheses) was measured by mixing exponential cultures of about 2 x 10s cells/ml in the ratio of 1 Hfr to 10 F- recipients. Cells were incubated at 37°C for 30 min, after which mating was interrupted. Recombinants were selected on media containing 15 pg chloramphenicol/ml and 125 pg tetracycline/ ml. Hfr donor was GY7236, (HfrJ2 Zeu: : Tn!?: generously provided by R. Devoret) and recipient cells were plasmid-harbouring ,JC10289 strains. The data in the Table are the recombination frequencies relative to the wild-typetaken to be 100.

Kowalczykowski et al., 1989). However, another mutation that was produced upstream from Gly204, at residue 199, does not seem to decrease recombination (Cazaux et al., 1991). All these data suggest that RecA protein possesses a site between residues 204 and 225 involved in the recombination pathway. In this paper we describe the in viva and in vitro properties of two mutants leading to the replacement of the original Gly204 with either alanine or valine. These changes were expected to modify the interactions that possibly involve this site by progressively increasing steric hindrance and hydrophobic properties of residue 204. Our data demonstrate that residue 204 is not only essential for RecA protease activity but also participates actively in recombination. The recA430 allele displays a minor change of amino acid residue (Gly+Ser) leading to a decreased interaction of RecA protein with the repressors without greatly affecting recombination (Devoret et al., 1983). In order t,o analyse the role of residue 204 in the various RecA functions, we introduced new, minor modifications to the orginal glycine residue by adding first one methyl group (Ala), then an ispropyl group (Val), thus providing an increase in the steric hindrance and the hydrophobic feature of the protein. In this case, the last t’wo bases of codon 204 were changed (Cazaux et al., 1991). Both mutations led to new restriction sites that allow easy screening. recA604 and recA605 mutated genes were then cloned into the BamHI site of pBR322.

Two-dimensional electrophoresis showed that the purified mutated proteins have the same structural properties as wild-type RecA (data not shown). Table 1 shows that both recA604 and recA605 mutants, like recA430, are sensitive to ultra-violet light (u.v.), recA605 being twice as sensitive as the other two, without reaching the null phenotype. This confirms that neither of the chemical groups added at position 204 inactivated the RecA protein. Induction of the SOS response after u.v.-irradiation demonstrated that the recA605 mutant, was as affected as recA430 in LexA-repressor cleavage, whereas the recA604 mutant had an intermediate capability to induce the SOS response (Table 1). Thus the presence of one methyl group on amino acid residue 204 appears to affect only moderately the coprotease activity of RecA protein. However. when residue 204 carries either a hydroxyl (RecA430) or an isopropyl group (RecA605), the interaction between RecA protein and LexA repressor seems to be strongly hindered. This result’ was confirmed by measuring the in vitro cleavage of LexA repressor (generous gift from Dr M. Schnarr), in the presence of u.v.-irradiated supercoiled DNA (data not shown). In addition, neither recA604 nor recA605 mutants were able to induce Weiglereactivation, and no mutants could be detected in the phage progeny produced by these mutants (data not shown). This absence of error-prone repair in the recA604 mutant while the SOS response is still induced could be explained by an inability of the

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0

5

IO 2040

60 00 120

0

5

IO 20 40 6080

120

Time (mm) RecA605

RecA604

Time (min)

603 4

is 40 E t :

30t

A<,

0

i-+-----1

p-----o/

,.-

.A

,,,...”

IO 20

. I / ;.P i

‘,’

A.....

30 40

C’ ,

50

,/‘

,

,

,

60 70

80

90

1

,

,

100110

,

I

120 130

Time (mm) (b)

Figure 1. DNA strand exchange assay. Wild-type and protein-promoted strand exchange mutant RecA measured by agarose gel electrophoresis using the procedure described by Riddles & Lehman (1985). The reaction mixture was as described (Menetski et al., 1990) except that 9 PM-ssDNA or dsDNA, 0% PM-SSB protein and 5 PM-RecA protein were used. Ml3 ssDNA was preincubated with SSB protein for 10 min at 37 “C then RecA protein and ATPyS were added (ATP hydrolysis unnecessary for intermediate formation: Menetski et al., 1990). The incubation was carried on for another 10 min and the strand-exchange reaction was initiated by the addition of Ml3 dsDNA linearized by EcoRI digestion. The reaction volume was 200 ~1. The reaction was stopped at the indicated times by adding SDS to 1 o/0 (w/v) and EDTA to 50 InM and left for 10 min at 37°C to deproteinize. DNA samples (20 ~1) were loaded on a 0+3% agarose gel. Electrophoresis was carried out in TAE buffer (40 mu-Tris-acetate (pH %O), 2 mM-EDTA) for 14 h at 2.0 V/cm at 4°C. Low voltage and temperature were used,

mutated protein to promote UmuD cleavage required for mutagenesis (Nohmi et al., 1988). This would support the proposal that UmuD cleavage could be more sensitive to a methylation at position 204 in RecA protein than LexA cleavage. These results confirm the essential role of residue 204 in the interaction of RecA with LexA repressor and UmuD protein. The recA605 mutant, like the recA430 mutant, was deficient in the induction of the SOS response but was more u.v.-sensitive. This raises the possibility that the recA605 mutant is affected in the recombination process. Conjugational homologous recombination and intrachromosomal recombination were measured in the presence of all three alleles (Table 1). recA605 is strongly affected in both pathways of recombination, without reaching a recA null phenotype. In contrast, recA604 behaves like recA430. This result correlates with the important role of recombination in bacterial survival (Calsou & Defais, 1985). The frequencies of recombinants in genetic crosses are similar in the last two mutants and wild-type bacteria. From these results: it can be inferred that residue 204 of RecA protein is required for an interaction involved in recombination, since the presence of one methyl group leads to a lower recombination ability and an isopropyl group prevents about 95% of recombination. In an attempt to elucidate what function of RecA protein is impaired in recA605 and to compare various defects due to the presence of either an isopropyl group or a methyl group on residue 204, we purified the two RecA mutant proteins that we created, as well as RecA430 and wild-type proteins, which were used as controls (Cox et al., 1981). The most commonly used model reflecting in viva genetic recombination is the DNA strand exchange bet,ween a circular ssDNA and the homologous linearized dsDNA. Several steps constitute the complete mechanism of DNA strand exchange (for reviews, see Howard-Flanders et al., 1987; Kowalczykowski, 1987; Cox & Lehman, 1987; Radding, 1988; Roca & Cox, 199(j), including binding of RecA protein to DNA, homologous pairing and DNA-dependent ATPase activity, related to formation of the presynaptic and synaptic complexes or to processive unwinding of duplex DNA.

respectively,

to eliminate

current-induced

gels and to prevent deproteinized,

heating

of the

joined molecules from

dissociating back into substrates at room temperature (Rosselli & Stasiak, 1990). The gels were photographed (a) and negative films were scanned by a double beam recording microdensitometer (MKIIIC, Joyce-Loebl and Co. Ltd, UK). 1, recombinational intermediates and

products;

2, Ml3 dsDNA;

3 Ml3 ssDNA. (b) Peak areas

were measured to obtain the relative amounts of recombination intermediates, which are the ratios between the sum of the peak areas corresponding to all intermediates DNA species and total DNA. -O-O-. RecAwt;

--.--*--, RecA604: . . .A. . . .A.

RecA430; ., RecA605.

-.-m-.-m-.-,

C. Cazaux and M. Defais

826 60

I

I

I

I

I

I 15

I 20

I 25

30

I 35

Time (min)

Figure 2. Homologous pairing assay (D-loop aasay). Joined molecule formation was measured by retention of complexes of linear ‘H-labelled Ml3 dsDNA and homologous ssDNA on nitrocellulose filters (Millipore HAWPO2500. pore size 045 pm) as described by Beattie et al. (1977). The standard reaction mixture contained 25 mi+Tris. HCI 1 mM-ATP, @4 PM-SSB protein. (pH 7.5), 10 mM-magnesium acetate, 3.7% (v/v) glycerol, 1 mnn-dithiothreitol, 11.0 pm-M13 ssDNA, 150 PM-linear Ml3 ds[3H]DNA and 6 PM-RecA protein. An ATP-regenerating system, consisting of 4.4 mM-phosphoenolpyruvate and 2 units pyruvate kinaae/ml, was added. The reaction was carried out at 37°C and initiated by the simultaneous addition of SSB and RecA proteins. At the times indicated, 25 ~1 portions were removed and the reactions terminated by adding SDS to @5% (w/v) and EDTA to 12 mm After 20 s, 1 ml of 10 x SSC (2 M-NaCl. @15 M-sodium citrate) was added and the mixture was kept on ice. Within 10 min the samples were filtered at approximately 3 ml/10 s through nitrocellulose filters that had been previously soaked with 10 x SSC and washed with 10 ml of 10 x SSC. The filters were dried and radioactivity quantitated by liquid scintillation. 100% single-stranded assimilation was assayed by heating a portion of the reaction mixture at 100°C for 6 min then cooling quickly on ice as previously described (Cox & Lehman, 1981). After addition of SDS, EDTA and 1 ml of 10 x SSC, this sample was filtered as before. Background retention was determined using the same procedure without heating. - 0 - 0 -. RecAwt; --a--@--, RecA430; -.-m-.-m-.-, RecA604; ....A....A...., RecA605. Recombination intermediates catalysed by wildtype RecA (RecAwt), RecA430, RecA604 and RecA605 proteins under the same conditions were followed as a function of time. The amount of DNA that entered the intermediates was quantified with agarose gels (Fig. 1). Following the appearance of recombination intermediates promoted by wild-type or mutated RecA proteins, a concomitant decrease of linear dsDNA occurred. RecA605 protein was able to promote only little strand exchange corresponding to its residual recombination capacity observed in viva. RecA604 protein appeared slightly more impaired in DNA strand exchange than RecA430 protein. The first step in the DNA strand exchange reaction is the presynaptic phase (for reviews, see Howard-Flanders et al., 1987; Cox & Lehman, 1987; Kowalczykowski, 1987; Radding, 1988; Rota & Cox, 1990). During this phase, in the presence of ATP, RecA protein polymerizes on ssDNA to form nucleoprotein filament. This complex is stabilized by the presence of single-stranded-DNA-binding (SSB) protein (Morrical & Cox, 1990). It has been

two suggested that RecA protein possesses DNA-binding sites, one devoted to ssDNA, the other allowing the formation of a ternary complex with dsDNA in the presence of ATP (HowardFlanders et al., 1984; Bryant et al., 1985; Tsang et aZ., 1985; Ikawa et al., 1989). It was also reported that RecA430 protein retained a functional ssDNA binding site, but lost the second site, which affects homologous pairing (Ikawa et al., 1989). The retardation on agarose gel of the nucleoprotein complex formed by RecA430 protein and ssDNA was slightly smaller than that observed in the presence of RecAwt (data not shown). Under the same conditions, RecA604 and RecA605 proteins were hardly more affected in this binding than was RecA430 protein and both mutant proteins behaved identically. This indicates that residue 204 is not part of the ssDNA-binding site and is not involved in the formation of the presynaptic nucleoprotein complex. Once the presynaptic filament is formed, the nucleoprotein searches for homology by non-specific binding to duplex DNA (for reviews, see Howard-Flanders et al., 1987; Cox & Lehman, 1987;

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-0.10 5 : E E 6 5! :: 8 c : $I Y D

-0.20

-

-0.30

-

-0.40

\

-0*50

-

-0.60

-

-0.70 0

15

20

25

l-2 nmol/min

30

Time (min)

Figure 3. Strand-exchange-related ATP hydrolysis. The DNA-dependent ATPase activities of the RecA proteins were measured according to the spectrophotometric method described by Panuska & Goldwait (1980) and modified by Kreuzer k Jongeneel (1983). The hydrolysis of ATP to ADP is linked to the oxydation of NADH to NAD+ by the combined action of pyruvate kinase and L-lactate dehydrogenase. Phosphoenol pyruvate is converted to pyruvate by pyruvate kinase when ADP is regenerated to ATP. Then pyruvate is transformed to L-lactate by L-lactic dehydrogenase upon oxidation of NADH to NAD+. The buffer compositions and the ATPase assays were described by Kowalczykowski & Krupp (1987) and Kowalczykowski et al. (1989), except that DNA and RecA concentrations were, respectively, 86 and 3 PM. The reaction buffer containing DNA was incubated for 3 min at 37°C before wild-type or mutant protein was added. The ATP hydrolysis was monitored by following the rate of reduction in absorbance at 346 nm on a Beckman DU54 spectrophotometer coupled with a VPC computer. The change of absorbance is linked to the oxidation of NADH and proportional to the ATP-hydrolysis rate. This rate is equal in nmol/min per ml to the absolute value of the slope of absorbance reduction with time (dA/dt), measured on the linear portion of the curve, divided by the NADH extinction coefficient, or in numerical terms: -dA/dt x @16 (Kreuzer & Jongeneel, 1983). It was calculated for the @5 ml vplume employed (values at the right). -, RecAwt; - -, RecA430; -.-, RecA604; ......, RecA605.

Kowalczykowski, 1987; Radding, 1988; Rota & Cox, 1990). When completely homologous molecules interact, RecA protein promotes the formation of an heteroduplex plectonemic joint by displacement from a free end of one strand of the duplex DNA to circular single-stranded the complementary molecule. This joint can be detected by the D-loop assay (Shibata et al., 1979). Figure 2 shows that wild-type and RecA604 proteins catalysed the formation of plectonemic joint with the same kinetics, showing that the second DNA-binding site of ReeA604 protein is not affected. Since RecA604 protein retains the ability to form the presynaptic complexes and plectonemic joints, but is impaired in strand exchange reaction, it must be affected in another step of this mechanism. When RecA605 protein catalysed the reaction, 80% of plectonemic joints were formed at a much slower rate. The maximum number of joint molecules was formed after five minutes in the presence of RecAwt or RecA604 protein, whereas RecA605 protein needed about 20 minutes to catalyse the reaction. It appears, then, that the presence of an isopropyl group at position 204 of the protein kinetically affects homologous pairing without greatly reducing the final concentration of joint molecules. Binding of RecA protein to ssDNA does not require ATP, although ATP does, however, increase

the affinity of the protein for its substrate. ATP hydrolysis leads to a low binding affinity of RecA protein to ssDNA. The ATPase activity associated with RecA protein seems to allow it to alternatively bind to ssDNA and dissociate from it (Bryant et al., 1985; Menetski & Kowalczykowski, 1985). ATP hydrolysis is also required during branch migration when one strand of duplex DNA is displaced by the single strand of the nucleoprotein filament (Cox & Lehman, 1981; Honigberg et al., 1985). The dsDNAdependent ATPase activity results in the unwinding of duplex DNA that is necessary for the formation of the plectonemic joints (Iwabuchi et al., 1983). Figure 3 shows that the ATPase activity of RecA protein involved in strand exchange was strongly reduced in all three mutants analysed. Nevertheless it still persists, even in the RecA605 mutant, which is the most affected. This correlates with the results presented in Figure 1, where a small amount of recombination intermediates was formed by the RecA605 protein, and in Table 1, where in viva recombination was strongly reduced in this mutant. Taken together, these data suggest that residue 204 belongs to a site of RecA protein that is involved in some way in the unwinding of dsDNA. The ssDNAdependent ATPase activity was not affected by recA430, recA604 or recA605 mutations (data not shown). Thus, the modifications introduced into the

828

C. Cazaux and M. Defais

protein do not interfere with the formation synaptic filaments, which confirms the

of pre-

above-

mentioned results. It can be concluded from these results that residue 204 of RecA protein modulates not only the proteolysis of LexA repressor and UmuD protein, but also recombination, where it appears to play an

essential role in the catalysis of helicase activity. However, it may seem surprising that such a reduced domain of RecA protein is involved in so many activities. Yu & Egelman (1990) have recently shown that RecA protein is organized into two domains and that ATP-dependent changes in domain-domain organization would be responsible for rotational motions (Egelman & Stasiak, 1988) and consequently for transfer of a DNA strand from one DNA molecule to another in recombination. We suggest that the site downstream from residue 204 could be related to the production of signals directing similar allosteric changes and modulating RecA activities, thus regulating SOS repair and/or recombination pathways in accordance with cellular needs. It can also be supposed, as an alternative hypothesis, that residue 204 is situated in the wide groove of the RecA helical coat around DNA that appears to play a primordial role both in the strandexchange model (Howard-Flanders et al., 1984) and in LexA cleavage (Hewat et al., 1991). The analysis of other mutants will be necessary to define more precisely the limit of this site and to understand more clearly the role of this domain. We are grateful for Drs N. P. Johnson and G. Villani for their helpful suggestions and critical comments on the manuscript. We thank H. Mazarguil for his help for synthesis of oligonucleotides. This work was partly supported by a grant) no. 8800497 from the Regional Council of Midi-Pyr6n6es. References Beattie, K. L., Wiegang, R. C. & Radding, C. M. (1977). Uptake of homologous single stranded fragments by superhelical DNA. J. Mol. Biol. 116, 783-803. Blanco, M., Levine, A. & Devoret, R. (1975). LexB: a new gene governing radiation sensitivity and lysogenic induction in Escherichia coli K-12. In Molecular Mechanisms joy Repair of DNA (Hanawalt, P. & Setlow, R. B., eds), pp. 379-382, Plenum Press, New York. Bolivar, F., Rodriguez, R. L., Greene, P. J., Crosa, J. H.. Betlach, M. C., Heyneker, H. L. & Boyer, H. W. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 2, 95-113. Bryant, F. R., Taylor, A. & Lehman, I. R. (1985). Interaction of the RecA protein of Escherichia coli with single-stranded DNA. J. Biol. Chem. 260, 1196-1202. Burckhardt, S. E., Woodgate, R., Sheuermann, R. H. & Echols, H. (1988). The UmuD mutagenesis protein of Escherichia coli: overproduction, purification and cleavage by RecA. Proc. Nat. Acad. Sci., U.S.A. 85, 1811-1815. Calsou, P. & Defais, M. (1985). Weigle reactivation and

mutagenesis of bacteriophage lambda in LsrA (DefJ mutants of E. coli K12. Mol. Gen. Genet. 201. 1162-I 165. Cazaux, C., Larminat, F. & Defais, M. (1991). Site-directed mutagenesis in the Escherichia coli recA gene. Biochimie, 73, 281-284. Clark, A. J. (1973). Recombination deficient mutants of E. coli and other bacteria. Annu. Rev. Genet. 71. 67-86. Clark, A. J. & Margulies, A. D. (1965). Isolation and characterization of recombination-deficient mutants of Escherichia coli K12. Proc. Nat. Acad. Sci., l/‘.S.A. 53, 451-459. Cox. M. M. & Lehman, I. R. (1981). RecA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc. Nat. Acad. Sci., U.S.A. 78, 3433-3437. Cox: AM.M. & Lehman, I. R. (1987). Enzymes of general recombination. Annu. Rev. Biochem. 56, 229-262. Cox. M. M., McEntee, K. & Lehman, I. R. (1981). A simple and rapid procedure for the large scale purification of the RecA protein of Escherichia coli. J. Biol.

Chem. 256, 4676-4678.

Csonka, L. N. & Clark, A. J. (1979). Deletions generated by the transposon TnlO in the Srl-RecA region of the Escherichia coli K-12 chromosome. Genetics, 93. 321-343. Devoret, R., Pierre, M. & Moreau, P. L. (1983). Prophage @80 is induced in Escherichia coli K12 recA430. Mol. Gen. Genet. 189, 199-206. Dutreix, M., Bailone, A. & Devoret, R. (1985). Efficiency of induction of prophage lambda mutants as a function of recA alleles. J. Bacterial. 161, 1080-1085. Dutreix, M., Moreau, P. L., Bailone, A., Galibert, F., Battista, J. R., Walker, G. C. & Devoret, R. (1989). New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidenrr for an additional role for RecA protein in UV-mutagenesis, J. Bacterial. 115, 2415-2423. Egelman, E. H. & Stasiak, A. (1988). Structure of helical RecA-DNA complexes. II. Local conformational changes visualized in bundles of RecA-ATP-y-S filaments. J. Mol. Biol. 200, 329-349. Hewat, E. A., Ruigrok, R. W. H. & DiCapua, E. (1991). Activation of RecA protein: the pitch of the helical complex with single-stranded DNA. EMBO J. 10, 2695-2698. Honigberg, S. M., Gonda, D. K., Flory, J. & Radding, C. M. (1985). The pairing activity of stable nucleoprotein filaments made from RecA protein. single-stranded DNA, and adenosine 5’.(y-thio) triphosphate, J. Biol. Chem. 260, 11845-I 1851. Howard-Flanders, P.. West, S. C., Rusche. *J. R. & Egelman, E. (1984). Molecular mechanisms of general genetic recombination: the DNA-binding sites of RecA protein. Cold Spring Harbor Symp. Quant. Biol. 49, 571-580. Howard-Flanders, I’.. West, S. (I., Cassuto. E.. Hahn. T. R. & Egelman, E. (1987). St,ructure of RrcA spiral filaments and t,heir role in homologous pairing and strand exchange in genetic recombination. In DXA Replication and Recombination (McMaken. R. & Kelly, T. .J.. eds), pp. 609-617. A. R. Liss. Inc.. New York. Ikawa, S., Kamiya, N. h Shibata, T. (1989). Defective and proficient proces’sive homologous pairing unwinding by the RecA430 mutant protein and intermediates of homologous pairing by RevA protein. .I. Biol. Chem. 264, 21167-21176.

Communications Iwabuchi, M., Shibata, T., Ohtani, T., Natori, M. & Ando, T. (1983). ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli RecA protein in the presence of topoisomerase. J. Biol. Chem. 258, 12394-12404. Kawashima, H., Horii, T., Ogawa, T. & Ogawa, H. (1984). Functional domains of E. coli RecA protein deduced from mutational sites in the gene. Mol. Gen. Genet.

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by N. Sternberg