Purification and characterization of Rad3 ATPase/DNA helicase from Saccharomyces cerevisiae

The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells containing overexpressed Rad3 protein. The DNA helicase can unwind duplex regions as short as 11 base pairs in a partially duplex circular DNA substrate and does so by a strictly processive mechanism. On partially duplex linear substrates, the enzyme has a strict 5'----3' polarity with respect to the single strand to which it binds. Nicked circular DNA is not utilized as a substrate, and the enzyme requires single-stranded gaps between 5 and 21 nucleotides long to unwind oligonucleotide fragments from partially duplex linear molecules. The enzyme also requires duplex regions at least 11 base pairs long when these are present at the ends of linear molecules. Rad3 DNA helicase activity is inhibited by the presence of ultraviolet-induced photoproducts in duplex regions of partially duplex circular molecules.     

Substrate specificity of the Rad3 ATPase/DNA helicase of Saccharomyces cerevisiae and binding of Rad3 protein to nucleic acids.

Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase which catalyzes the unwinding of DNA.DNA duplexes. In the present studies we have demonstrated that the purified enzyme additionally catalyzes the displacement of RNA fragments annealed to complementary DNA. Quantitative comparisons using otherwise identical partially duplex DNA.DNA and DNA.RNA substrates indicate a significant preference for the latter. Competition for ATPase or DNA helicase activity by various homopolymers suggests that Rad3 protein does not discriminate between ribonucleotide and deoxyribonucleotide homopolymers with respect to binding. However, neither single-stranded RNA nor various ribonucleotide homopolymers supported the hydrolysis of nucleoside 5'-triphosphates. Additionally, Rad3 protein was unable to catalyze the displacement of oligo(dA) annealed to poly(U), suggesting that the catalytic domain of the enzyme is exquisitely sensitive to chemical and/or or conformational differences between DNA and RNA. Hence, it appears that Rad3 protein is not an RNA helicase.     

Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange.

Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.     

Formation of joint DNA molecules by two eukaryotic strand exchange proteins does not require melting of a DNA duplex

We have examined whether DNA strand exchange activities from nuclear extracts of HeLa cells or Drosophila melanogaster embryos have detectable helicase or melting activities. The partially purified recombinases have been shown to recognize homologous single strand and double strand DNA molecules and form joint molecules in a DNA strand exchange reaction. The joint molecule product consists of a linear duplex joined at one end by a region of DNA heteroduplex to a homologous single strand circular DNA. Using two different partially duplex helicase substrates, we are unable to detect any melting of duplex regions under conditions that promote joint molecule formation. One substrate consists of a 32P-labeled oligonucleotide 20 or 30 bases long annealed to M13mp18 circular single strand DNA. The second substrate consists of a linear single strand region flanked at each end by short duplex regions. We observe that even in the presence of excess recombinase protein or after prolonged incubation no helicase activity is apparent. Control experiments rule out the possibility that a helicase is masked by reannealing of displaced single strand fragments. Based on these findings and other data, we conclude that the human and D. melanogaster recombinases recognize and pair homologous sequences without significant melting of duplex DNA prior to strand exchange.     

Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity.

A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells. At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity. At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity. Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA. The addition of E. coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase. One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase. Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity. The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction. The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment. Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.     

Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein.

Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro. In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome. When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion. However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region. With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%). On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate. These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis

Brh2 is the Ustilago maydis ortholog of the BRCA2 tumor suppressor. It functions in repair of DNA by homologous recombination by controlling the action of Rad51. A critical aspect in the control appears to be the recruitment of Rad51 to single-stranded DNA regions exposed as lesions after damage or following a disturbance in DNA synthesis. In previous experimentation, Brh2 was shown to nucleate formation of the Rad51 nucleoprotein filament that becomes the active element in promoting homologous pairing and DNA strand exchange. Nucleation was found to be initiated at junctions of double-stranded and single-stranded DNA. Here we investigated the DNA binding specificity of Brh2 in more detail using oligonucleotide substrates. We observed that Brh2 prefers partially duplex structures with single-stranded branches, flaps, or D-loops. We found also that Brh2 has an inherent ability to promote DNA annealing and strand exchange reactions on free as well as RPA-coated substrates. Unlike Rad51, Brh2 was able to promote DNA strand exchange when preincubated with double-stranded DNA. These findings raise the notion that Brh2 may have roles in homologous recombination beyond the previously established Rad51 mediator activity.     

Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.     

Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein

Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. Herein, we address the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. We show that Saccharomyces cerevisiae Rad54 exerts distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. We suggest that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination.     

Characterization of the bacteriophage T4 gene 41 DNA helicase

The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro. We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L. L., and Nossal, N. G. (1982) J. biol. Chem. 257, 12426-12434). The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein. We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork. Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template). This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork. As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished. However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs). These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein. A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity). In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.     

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.     

Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro.

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.     

A DNA helicase induced by herpes simplex virus type 1.

We have identified and partially purified a DNA-dependent ATPase that is present specifically in herpes simplex virus type 1-infected Vero cells. The enzyme which has a molecular weight of approximately 440,000 differs from the comparable host enzyme in its elution from phosphocellulose columns and in its nucleoside triphosphate specificity. The partially purified DNA-dependent ATPase is also a DNA helicase that couples ATP or GTP hydrolysis to the displacement of an oligonucleotide annealed to M13 single-stranded DNA. The enzyme requires a 3' single-stranded tail on the duplex substrate, suggesting that the polarity of unwinding is 5'----3' relative to the M13 DNA. The herpes specific DNA helicase may therefore translocate on the lagging strand in the semidiscontinuous replication of the herpes virus 1 genome.     

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.     

Simian virus 40 large T antigen DNA helicase. Characterization of the ATPase-dependent DNA unwinding activity and its substrate requirements

The ATPase of SV40 large T antigen (T antigen) which is essential for the replication of SV40 minichromosomes was recently shown to be functionally related to a newly discovered DNA helicase activity. The T antigen helicase unwinds DNA duplices of several kilobase pairs in a reaction depending on the presence of hydrolyzable ribo- or deoxyribonucleoside triphosphates. The in vitro rate of movement through duplex DNA was found to be about 100 base pairs/min at 37 degrees C. For DNA unwinding, T antigen requires a 3'-single strand extension of a partially double-stranded substrate and invades the double strand section processively, in a 3' to 5' direction. The minimum length of the single-stranded tail was determined to be less than 5 nucleotides. Unwinding studies in the presence of the Escherichia coli single strand-specific DNA-binding protein and competition experiments indicate that T antigen helicase binds preferentially at the single-stranded/double-stranded DNA junction. This DNA structure is therefore proposed to serve as an entry site for the T antigen helicase. Previously reported data suggest that T antigen is the replicative helicase of the SV40 minichromosome. The results presented here are consistent with these findings and imply that T antigen migrates actively and processively along the template for the leading strand.     

Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.     

DNA strand exchange catalyzed by proteins from vaccinia virus-infected cells.

Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex. The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product. Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps. The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa. The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+. One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing. As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors. Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail. The recovery of these structures shows that the enzyme catalyzes true strand exchange. There is also a unique polarity to the strand exchange reaction. The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand. Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product. Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.     

Pestivirus NS3 (p80) protein possesses RNA helicase activity.

The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed.     

Rad51 gain-of-function mutants that exhibit high affinity DNA binding cause DNA damage sensitivity in the absence of Srs2

We previously identified several rad51 gain-of-function alleles that partially suppress the requirement for RAD55 and RAD57 in DNA repair. To gain further insight into the mechanism of action of these alleles, we compared the activities of Rad51-V328A, Rad51-P339S and Rad51-I345T with wild-type Rad51, for DNA binding, filament stability, strand exchange and interaction with the antirecombinase helicase, Srs2. These alleles were chosen because they show the highest activity in suppression of ionizing radiation sensitivity of the rad57 mutant, and Val 328 and Ile 345 are conserved in the human Rad51 protein. All three mutant proteins exhibited higher affinity for single-stranded DNA (ssDNA) and showed more robust strand exchange activity with oligonucleotide substrates than wild-type Rad51, with the Rad51-I345T and Rad51-V328A proteins displaying higher activity than Rad51-P339S. However, the Srs2 antirecombinase was able to disrupt Rad51–ssDNA complexes formed with all the mutant proteins. In vivo, the rad51-I345T mutant strain exhibited high resistance to methyl methane sulfonate that was dependent on functional SRS2. These results suggest the Srs2 translocase is able to disrupt Rad51–ssDNA complexes at stalled replication forks, but in the absence of Srs2 the enhanced DNA binding of the Rad51-I345T protein is detrimental to cell survival.