Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance

Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol alpha-primase. In addition, a delayed senescence phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance.     

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.     

Fission yeast Taz1 and RPA are synergistically required to prevent rapid telomere loss

The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast.

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein

Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.     

RPA provides checkpoint-independent cell cycle arrest and prevents recombination at uncapped telomeres of Saccharomyces cerevisiae

Replication Protein A (RPA) is an evolutionary conserved essential complex with single-stranded DNA binding properties that has been implicated in numerous DNA transactions. At damaged telomeres, Saccharomyces cerevisiae RPA recruits the Mec1–Ddc2 module of the DNA damage checkpoint network, its only known function in DNA damage signaling. Here, we describe rfa1 mutants (rfa1-1, rfa1-9, rfa1-10, rfa1-11 and rfa1-12) that are proficient in this checkpoint but nevertheless exhibit deregulation of cell cycle control upon telomere uncapping induced by the cdc13-1 mutation. Overriding of this damage-induced checkpoint-independent cell cycle block in the rfa1 mutants was suppressed following genetic inactivation of either TEL1 or EST2/telomerase. Altogether, our results suggest that a previously non-suspected function of RPA is to block cell cycle progression upon telomere uncapping using a yet unidentified pathway that functions in a Mec1–Ddc2-independent manner. We propose that in the rfa1 mutants, ill-masking of uncapped telomeres provokes inappropriate access of Tel1 and inappropriate functioning of telomerase, which, by yet unknown mechanisms, allows cell division to take place in spite of the block established by the DNA damage checkpoint. In the present study, we also observed that upon telomere uncapping, rfa1-12, but not the other studied rfa1 mutants, triggered telomeric recombination in the presence of functional telomerase. In conclusion, the present study identifies a novel pathway of telomere end protection that utilizes a previously unsuspected function of RPA at the telomeres.     

Studies of the Interaction between Rad52 Protein and the Yeast Single-Stranded DNA Binding Protein RPA

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729–10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

Regulation of Telomere Length by Checkpoint Genes in Schizosaccharomyces pombe

We have studied telomere length in Schizosaccharomyces pombe strains carrying mutations affecting cell cycle checkpoints, DNA repair, and regulation of the Cdc2 protein kinase. Telomere shortening was found in rad1, rad3, rad17, and rad26 mutants. Telomere lengths in previously characterized rad1 mutants paralleled the replication checkpoint proficiency of those mutants. In contrast, rad9, chk1, hus1, and cds1 mutants had intact telomeres. No difference in telomere length was seen in mutants affected in the regulation of Cdc2, whereas some of the DNA repair mutants examined had slightly longer telomeres than did the wild type. Overexpression of the rad1⁺ gene caused telomeres to elongate slightly. The kinetics of telomere shortening was monitored by following telomere length after disruption of the rad1⁺ gene; the rate was ~1 nucleotide per generation. Wild-type telomere length could be restored by reintroduction of the wild-type rad1⁺ gene. Expression of the Saccharomyces cerevisiae RCK1 protein kinase gene, which suppresses the radiation and hydroxyurea sensitivity of Sz. pombe checkpoint mutants, was able to attenuate telomere shortening in rad1 mutant cells and to increase telomere length in a wild-type background. The functional effects of telomere shortening in rad1 mutants were assayed by measuring loss of a linear and a circular minichromosome. A minor increase in loss rate was seen with the linear minichromosome, and an even smaller difference compared with wild-type was detected with the circular plasmid.     

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.     

RPA prevents G‐rich structure formation at lagging‐strand telomeres to allow maintenance of chromosome ends

Replication protein A (RPA) is a highly conserved heterotrimeric single‐stranded DNA‐binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1‐D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging‐strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G‐quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G‐quadruplex. We propose that RPA prevents the formation of G‐quadruplex structures at lagging‐strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.     

Repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in the fission yeast Schizosaccharomyces pombe

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6-4 photoproducts) in wild-type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13Δ, rad15 and rad16Δ, which are the S. pombe homologues of the excision-defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2Δ, also a presumed excision-defective mutant, behaves similarly. radS and rad9δ, which belong to different phenotypic groups, repair lesions at the same rate as wild-type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.     

The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.     

Rsp5, a ubiquitin-protein ligase, is involved in degradation of the single-stranded-DNA binding protein rfa1 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, RAD1 and RAD52 are required for alternate pathways of mitotic recombination. Double-mutant strains exhibit a synergistic interaction that decreases direct repeat recombination rates dramatically. A mutation in RFA1, the largest subunit of a single-stranded DNA-binding protein complex (RP-A), suppresses the recombination deficiency of rad1 rad52 strains (J. Smith and R. Rothstein, Mol. Cell. Biol. 15:1632-1641, 1995). Previously, we hypothesized that this mutation, rfa1-D228Y, causes an increase in recombinogenic lesions as well as the activation of a RAD52-independent recombination pathway. To identify gene(s) acting in this pathway, temperature-sensitive (ts) mutations were screened for those that decrease recombination levels in a rad1 rad52 rfa1-D228Y strain. Three mutants were isolated. Each segregates as a single recessive gene. Two are allelic to RSP5, which encodes an essential ubiquitin-protein ligase. One allele, rsp5-25, contains two mutations within its open reading frame. The first mutation does not alter the amino acid sequence of Rsp5, but it decreases the amount of full-length protein in vivo. The second mutation results in the substitution of a tryptophan with a leucine residue in the ubiquitination domain. In rsp5-25 mutants, the UV sensitivity of rfa1-D228Y is suppressed to the same level as in strains overexpressing Rfa1-D228Y. Measurement of the relative rate of protein turnover demonstrated that the half-life of Rfa1-D228Y in rsp5-25 mutants was extended to 65 min compared to a 35-min half-life in wild-type strains. We propose that Rsp5 is involved in the degradation of Rfa1 linking ubiquitination with the replication-recombination machinery.     

A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endonuclease. Besides isolating a number of new alleles in known rad genes, we identified a novel allele of the RFA1 gene, rfa1-44, which encodes the large subunit of the heterotrimeric yeast single-stranded DNA-binding protein RPA. Characterization of rfa1-44 revealed that it is, like members of the RAD52 epistasis group, sensitive to X rays, high doses of UV, and HO-induced DSBs. In addition, rfa1-44 shows a reduced ability to undergo sporulation and HO-induced gene conversion. The mutation was mapped to a single-base substitution resulting in an aspartate at amino acid residue 77 instead of glycine. Moreover, all radiation sensitivities and repair defects of rfa1-44 are suppressed by RAD52 in a dose-dependent manner, and one RAD52 mutant allele, rad52-34, displays nonallelic noncomplementation when crossed with rfa1-44. Presented is a model accounting for this genetic interaction in which Rfa1, in a complex with Rad52, serves to assemble other proteins of the recombination-repair machinery at the site of DSBs and other kinds of DNA damage. We believe that our findings and those of J. Smith and R. Rothstein (Mol. Cell. Biol. 15:1632-1641, 1995) are the first in vivo demonstrations of the involvement of a eukaryotic single-stranded binding protein in recombination and repair processes.     

Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination.

A new Schizosaccharomyces pombe mutant (rad32) which is sensitive to gamma and UV irradiation is described. Pulsed field gel electrophoresis of DNA from irradiated cells indicates that the rad32 mutant, in comparison to wild type cells, has decreased ability to repair DNA double strand breaks. The mutant also undergoes decreased meiotic recombination and displays reduced stability of minichromosomes. The rad32 gene has been cloned by complementation of the UV sensitive phenotype. The gene, which is not essential for cell viability and is expressed at a moderate level in mitotically dividing cells, has significant homology to the meiotic recombination gene MRE11 of Saccharomyces cerevisiae. Epistasis analysis indicates that rad32 functions in a pathway which includes the rhp51 gene (the S.pombe homologue to S.cerevisiae RAD51) and that cells deleted for the rad32 gene in conjunction with either the rad3 deletion (a G2 checkpoint mutation) or the rad2 deletion (a chromosome stability and potential nucleotide excision repair mutation) are not viable.     

Genome stability is guarded by yeast Rtt105 through multiple mechanisms

Ty1 mobile DNA element is the most abundant and mutagenic retrotransposon present in the genome of the budding yeast Saccharomyces cerevisiae. Protein regulator of Ty1 transposition 105 (Rtt105) associates with large subunit of RPA and facilitates its loading onto a single-stranded DNA at replication forks. Here, we dissect the role of RTT105 in the maintenance of genome stability under normal conditions and upon various replication stresses through multiple genetic analyses. RTT105 is essential for viability in cells experiencing replication problems and in cells lacking functional S-phase checkpoints and DNA repair pathways involving homologous recombination. Our genetic analyses also indicate that RTT105 is crucial when cohesion is affected and is required for the establishment of normal heterochromatic structures. Moreover, RTT105 plays a role in telomere maintenance as its function is important for the telomere elongation phenotype resulting from the Est1 tethering to telomeres. Genetic analyses indicate that rtt105Δ affects the growth of several rfa1 mutants but does not aggravate their telomere length defects. Analysis of the phenotypes of rtt105Δ cells expressing NLS-Rfa1 fusion protein reveals that RTT105 safeguards genome stability through its role in RPA nuclear import but also by directly affecting RPA function in genome stability maintenance during replication.     

Role of Saccharomyces single-stranded DNA-binding protein RPA in the strand invasion step of double-strand break repair

The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.     

Critical Functions of Rpa3/Ssb3 in S-Phase DNA Damage Responses in Fission Yeast

Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)–binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11^Rpa1 and Rad22^Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.     

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.