Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae

In wild-type diploid cells, heteroallelic recombination between his4A and his4C alleles leads mostly to His+ gene conversions that have a parental configuration of flanking markers, but approximately 22% of recombinants have associated reciprocal crossovers. In rad52 strains, gene conversion is reduced 75-fold and the majority of His+ recombinants are crossover associated, with the largest class being half-crossovers in which the other participating chromatid is lost. We report that UV irradiating rad52 cells results in an increase in overall recombination frequency, comparable to increases induced in wild-type (WT) cells, and surprisingly results in a pattern of recombination products quite similar to RAD52 cells: gene conversion without exchange is favored, and the number of 2n - 1 events is markedly reduced. Both spontaneous and UV-induced RAD52-independent recombination depends strongly on Rad50, whereas rad50 has no effect in cells restored to RAD52. The high level of noncrossover gene conversion outcomes in UV-induced rad52 cells depends on Rad51, but not on Rad59. Those outcomes also rely on the UV-inducible kinase Dun1 and Dun1's target, the repressor Crt1, whereas gene conversion events arising spontaneously depend on Rad59 and Crt1. Thus, there are at least two Rad52-independent recombination pathways in budding yeast.     

A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.

With the use of an intrachromosomal inverted repeat as a recombination reporter, we have shown that mitotic recombination is dependent on the RAD52 gene, but reduced only fivefold by mutation of RAD51. RAD59, a component of the RAD51-independent pathway, was identified previously by screening for mutations that reduced inverted-repeat recombination in a rad51 strain. Here we describe a rad52 mutation, rad52R70K, that also reduced recombination synergistically in a rad51 background. The phenotype of the rad52R70K strain, which includes weak gamma-ray sensitivity, a fourfold reduction in the rate of inverted-repeat recombination, elevated allelic recombination, sporulation proficiency, and a reduction in the efficiency of mating-type switching and single-strand annealing, was similar to that observed for deletion of the RAD59 gene. However, rad52R70K rad59 double mutants showed synergistic defects in ionizing radiation resistance, sporulation, and mating-type switching. These results suggest that Rad52 and Rad59 have partially overlapping functions and that Rad59 can substitute for this function of Rad52 in a RAD51 rad52R70K strain.     

Rad51-independent interchromosomal double-strand break repair by gene conversion requires Rad52 but not Rad55, Rad57, or Dmc1

Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.     

Genetic and Molecular Analysis of Recombination Events in Saccharomyces Cerevisiae Occurring in the Presence of the Hyper-Recombination Mutation Hpr1

The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion. The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype. The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved. hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5. This result suggests that different intrachromosomal recombination processes are affected in each mutant strain. We propose that HPR1 may function to inhibit intrachromatid crossovers.     

The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51.     

The Rad52 homologs Rad22 and Rti1 of Schizosaccharomyces pombe are not essential for meiotic interhomolog recombination, but are required for meiotic intrachromosomal recombination and mating-type-related DNA repair

Proteins of the RAD52 epistasis group play an essential role in repair of some types of DNA damage and genetic recombination. In Schizosaccharomyces pombe, Rad22 (a Rad52 ortholog) has been shown to be as necessary for repair and recombination events during vegetative growth as its Saccharomyces cerevisiae counterpart. This finding contrasts with previous reports where, due to suppressor mutations in the fbh1 gene, rad22 mutants did not display a severe defect. We have analyzed the roles of Rad22 and Rti1, another Rad52 homolog, during meiotic recombination and meiosis in general. Both proteins play an important role in spore viability. During meiotic prophase I, they partially colocalize and partially localize to Rad51 foci and linear elements. Genetic analysis showed that meiotic interchromosomal crossover and conversion events were unexpectedly not much affected by deletion of either or both genes. A strong decrease of intrachromosomal recombination assayed by a gene duplication construct was observed. Therefore, we propose that the most important function of Rad22 and Rti1 in S. pombe meiosis is repair of double-strand breaks with involvement of the sister chromatids. In addition, a novel mating-type-related repair function of Rad22 specific to meiosis and spore germination is described.     

Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.     

Effects of tumor-associated mutations on Rad54 functions

Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous recombination and DNA double strand break repair. Rad54 belongs to the Snf2/Swi2 protein family, and it possesses a robust DNA-dependent ATPase activity, uses free energy from ATP hydrolysis to supercoil DNA, and cooperates with the Rad51 recombinase in DNA joint formation. There are two RAD54-homologous genes in human cells, hRAD54 and RAD54B. Mutations in these human genes have been found in tumors. These tumor-associated mutations map to conserved regions of the hRad54 and hRad54B proteins. Here we introduced the equivalent mutations into the Saccharomyces cerevisiae RAD54 gene in an effort to examine the functional consequences of these gene changes. One mutant, rad54 G484R, showed sensitivity to DNA-damaging agents and reduced homologous recombination rates, indicating a loss of function. Even though the purified rad54 G484R mutant protein retained the ability to bind DNA and interact with Rad51, it was nearly devoid of ATPase activity and was similarly defective in DNA supercoiling and D-loop formation. Two other mutants, rad54 N616S and rad54 D442Y, were not sensitive to genotoxic agents and behaved like the wild type allele in homologous recombination assays. Consistent with the mild phenotype associated with the rad54 N616S allele, its encoded protein was similar to wild type Rad54 protein in biochemical attributes. Because dysfunctional homologous recombination gives rise to genome instability, our results are consistent with the premise that tumor-associated mutations in hRad54 and Rad54B could contribute to the tumor phenotype or enhance the genome instability seen in tumor cells.     

Interaction between Ustilago Maydis Rec2 and Rad51 Genes in DNA Repair and Mitotic Recombination

A gene encoding a Ustilago maydis Rad51 orthologue has been isolated. rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and γ radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.     

Characterization of the Roles of the Saccharomyces Cerevisiae Rad54 Gene and a Homologue of Rad54, Rdh54/Tid1, in Mitosis and Meiosis

The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDH54/TID1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of RAD54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.     

Interaction with Rad51 is indispensable for recombination mediator function of Rad52

In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409-420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52delta409-412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52delta409-412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination.     

Multiple Pathways for Homologous Recombination in Saccharomyces Cerevisiae

The genes in the RAD52 epistasis group of Saccharomyces cerevisiae are necessary for most mitotic and meiotic recombination events. Using an intrachromosomal inverted-repeat assay, we previously demonstrated that mitotic recombination of this substrate is dependent upon the RAD52 gene. In the present study the requirement for other genes in this epistasis group for recombination of inverted repeats has been analyzed, and double and triple mutant strains were examined for their epistatic relationships. The majority of recombination events are mediated by a RAD51-dependent pathway, where the RAD54, RAD55 and RAD57 genes function downstream of RAD51. Cells mutated in RAD55 or RAD57 as well as double mutants are cold-sensitive for inverted-repeat recombination, whereas a rad51 rad55 rad57 triple mutant is not. The RAD1 gene is not required for inverted-repeat recombination but is able to process spontaneous DNA lesions to produce recombinant products in the absence of RAD51. Furthermore, there is still considerably more recombination in rad1 rad51 mutants than in rad52 mutants, indicating the presence of another, as yet unidentified, recombination pathway.     

RAD51-independent inverted-repeat recombination by a strand-annealing mechanism

Recombination between inverted repeats is RAD52 dependent, but reduced only modestly in the rad51Δ mutant. RAD59 is required for RAD51-independent inverted-repeat recombination, but no clear mechanism for how recombination occurs in the absence of RAD51 has emerged. Because Rad59 is thought to function as an accessory factor for the single-strand annealing activity of Rad52 one possible mechanism for spontaneous recombination could be by strand annealing between repeats at a stalled replication fork. Here we demonstrate the importance of the Rad52 single-strand annealing activity for generating recombinants by showing suppression of the rad52Δ, rad51Δ rad52Δ and rad52Δ rad59Δ inverted-repeat recombination defects by the rfa1-D228Y mutation. In addition, formation of recombinants in the rad51Δ mutant was sensitive to the distance between the inverted repeats, consistent with a replication-based mechanism. Deletion of RAD5 or RAD18, which are required for error-free post-replication repair, reduced the recombination rate in the rad59Δ mutant, but not in wild type. These data are consistent with RAD51-independent recombinants arising by a faulty template switch mechanism that is distinct from nascent strand template switching.     

Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.     

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

The RAD1 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RAD1 gene on genetic recombination. Mitotic intrachromosomal and interchromosomal recombination in RAD+, rad1, rad52, and other rad mutant strains was examined. The rad1 deletion mutation and some rad1 point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the rad1 rad52 double mutant, indicating that the RAD1 and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RAD1 gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the rad1 delta mutation did lower the frequency of integration of linear plasmids and DNA fragments into homologous genomic sequences. We suggest that RAD1 plays a role in recombination after the formation of the recombinogenic substrate.     

Unrepaired Heteroduplex DNA in Saccharomyces Cerevisiae Is Decreased in Rad1 Rad52-Independent Recombination

A direct repeat recombination assay between SUP4 heteroalleles detects unrepaired heteroduplex DNA (hDNA) as sectored colonies. The frequency of unrepaired heteroduplex is dependent on the mismatch and is highest in a construct that generates C:C or G:G mispairs and lowest in one that generates T:G or C:A mispairs. In addition, unrepaired hDNA increases for all mismatches tested in pms1 mismatch repair-deficient strains. These results support the notion that hDNA is formed across the SUP4 repeats during the recombination event and is then subject to mismatch repair. The effects of various repair and recombination defective mutations on this assay were examined. Unrepaired heteroduplex increases significantly only in rad52 mutant strains. In addition, direct repeat recombination is reduced 2-fold in rad52 mutant strains, while in rad51, rad54, rad55 and rad57 mutants direct repeat recombination is increased 3-4-fold. Mutations in the excision repair gene, RAD1, do not affect the frequency of direct repeat recombination. However, the level of unrepaired heteroduplex is slightly decreased in rad1 mutant strains. Similar to previous studies, rad1 rad52 double mutants show a synergistic reduction in direct repeat recombination (35-fold). Interestingly, unrepaired heteroduplex is reduced 4-fold in the double mutants. Experiments with shortened repeats suggest that the reduction in unrepaired heteroduplex is due to decreased hDNA tract length in the double mutant strain.     

Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.     

Rad51 protein from the thermotolerant yeast Pichia angusta as a typical but thermodependent member of the Rad51 family

The Rad51 protein from the methylotrophic yeast Pichia angusta (Rad51(Pa)) of the taxonomic complex Hansenula polymorpha is a homolog of the RecA-RadA-Rad51 protein superfamily, which promotes homologous recombination and recombination repair in prokaryotes and eukaryotes. We cloned the RAD51 gene from the cDNA library of the thermotolerant P. angusta strain BKM Y1397. Induction of this gene in a rad51-deficient Saccharomyces cerevisiae strain partially complemented the survival rate after ionizing radiation. Purified Rad51(Pa) protein exhibited properties typical of the superfamily, including the stoichiometry of binding to single-stranded DNA (ssDNA) (one protomer of Rad51(Pa) per 3 nucleotides) and DNA specificity for ssDNA-dependent ATP hydrolysis [poly(dC) > poly(dT) > phiX174 ssDNA > poly(dA) > double-stranded M13 DNA]. An inefficient ATPase and very low cooperativity for ATP interaction position Rad51(Pa) closer to Rad51 than to RecA. Judging by thermoinactivation, Rad51(Pa) alone was 20-fold more thermostable at 37 degrees C than its S. cerevisiae homolog (Rad51(Sc)). Moreover, it maintained ssDNA-dependent ATPase and DNA transferase activities up to 52 to 54 degrees C, whereas Rad51(Sc) was completely inactive at 47 degrees C. A quick nucleation and an efficient final-product formation in the strand exchange reaction promoted by Rad51(Pa) occurred only at temperatures above 42 degrees C. These reaction characteristics suggest that Rad51(Pa) is dependent on high temperatures for activity.     

Yeast Intrachromosomal Recombination: Long Gene Conversion Tracts Are Preferentially Associated with Reciprocal Exchange and Require the Rad1 and Rad3 Gene Products

A yeast intrachromosomal recombination system based on an inverted repeat has been designed to examine mitotic gene conversion tract length and the association of crossing over with gene conversion as a function of the conversion tract length. Short conversion tracts are found to be preferentially noncrossover while conversion tracts longer than 1.16 kb show a 50% association with crossover. Mutation in the excision repair gene RAD1 leads to a reduction in conversion tracts of at least 1.16 kb and a reduction in crossovers associated with conversion, regardless of the length of the conversion tract. Mutation in the excision repair gene RAD3, which encodes a DNA helicase, also leads to a reduction in conversion tracts of at least 1.16 kb, but has no effect on the frequency of associated crossovers. The roles of RAD1 and RAD3 in recombination are discussed.     

DNA annealing mediated by Rad52 and Rad59 proteins

In the budding yeast Saccharomyces cerevisiae, the RAD52 gene is essential for all homologous recombination events and its homologue, the RAD59 gene, is important for those that occur independently of RAD51. Both Rad52 and Rad59 proteins can anneal complementary single-stranded (ss) DNA. We quantitatively examined the ssDNA annealing activity of Rad52 and Rad59 proteins and found significant differences in their biochemical properties. First, and most importantly, they differ in their ability to anneal ssDNA that is complexed with replication protein A (RPA). Rad52 can anneal an RPA-ssDNA complex, but Rad59 cannot. Second, Rad59-promoted DNA annealing follows first-order reaction kinetics, whereas Rad52-promoted annealing follows second-order reaction kinetics. Last, Rad59 enhances Rad52-mediated DNA annealing at increased NaCl concentrations, both in the absence and presence of RPA. These results suggest that Rad59 performs different functions in the recombination process, and should be more accurately viewed as a Rad52 paralogue.