The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression ofRADS1 or RADS2. Virtually complete suppression is provided by the simultaneous overexpression ofRADSI and RAD52. This suppression occurs at 23°C, where these mutants are more sensitive to x-rays, as well as at 30°C and 36°C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad5l and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.The RADSS and RAD57 genes of Saccharomyces cerevisiae belong to the RADS2 epistasis group, a group of genes (RAD5O-57, MRE2, MREJJ, XRS2, and RFAlt) whose products have been implicated in the recombinational repair of DNA double-strand breaks (DSBs) (for review see refs. 3-5). As a means to better understand the participants and mechanisms involved in the repair of DSBs, we devised a genetic screen designed to identify mutants unable to perform the recombinational repair of a targeted DSB and thereby identified a number of alleles of genes in the RAD52 epistasis group (2). Further studies of some of these mutants, specifically radS5 and rad57 mutants, suggested that there might be interactions between the gene products of certain members of this group. We have characterized these interactions in order to gain insight into the molecular mechanisms that mediate DSB repair in yeast.Lovett and Mortimer (6) reported that a rad55 null mutant was more sensitive to x-rays at 23°C than at 36°C. This result was striking in that cold sensitivity is usually observed with missense alleles and not deletion alleles. Cold sensitivity is, however, a property often associated with proteins composed of multiple subunits or large multiprotein complexes (7,8), and the authors suggested that the Rad55 protein might participate in some sort of higher-order complex responsible for the repair of x-ray-induced damage (6). Other members of the RAD52 epistasis group would be logical candidates for participation in such a complex. Indeed, interactions between Rad5l and Rad52 and between Rad5l and itself have been identified (9-11), and a Rad52-Rad52 interaction has been inferred (10, 12). There is also genetic evidence that an interaction between the RAD52 and RFAJ gene products is involved in the recombinational repair of DSBs (1, 2). Here we provide evidence for the existence of interactions among Rad5l, Rad52, Rad55, and Rad57 that affect recombination and repair, and we show that some of these are direct physical interactions. We propose that these four proteins together with RPA constitute part of aThe publication costs of this article were defrayed in part ...

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.The yeast single-stranded DNA binding protein replication protein A (RPA) is a multisubunit complex containing three polypeptides of 70, 30, and 14 kDa. This heterotrimeric structure is conserved across all eukaryotic species where the protein is found. In Saccharomyces cerevisiae, deletion of any one of the three subunits is lethal (3), a reflection of the critical role the protein complex plays in DNA replication. RPA's role in yeast is not limited to replication, however; it participates in repair and recombination as well. RPA's involvement in yeast recombination is revealed in biochemical studies of Rad51, which show that RPA is required for the Rad51-catalyzed formation of both joint molecules and fully exchanged products from single-stranded circular DNA and linear doublestranded DNA with an overhanging complementary end (31,38,39).Genetic analysis of yeast has underscored the importance of RPA in recombination, as mutations in the gene RFA1, encoding the large (70-kDa) subunit, affect recombination ability (8, 24, 37). One of these mutations, the rfa1-44 allele, results in a 390-fold reduction in a recombination assay that is based on the recombinational repair of HO-endonuclease-induced double-stranded breaks (DSBs) (8). In fact, the rfa1-44 mutant strain is approximately as deficient in its ability to repair a DSB-whether induced by the action of HO endonuclease or by exposure to X rays-as the rad55 and rad57 mutants tested (8,13,14). A further indication of the...

The structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein. The labeled oligonucleotide hybridized specifically to the A clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene. Approximately 1400 base pairs of dinA sequence were determined. The predicted amino-terminal sequence of d&A demonstrated that this gene encoded DNA polymerase II. Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the d&A promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene. These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon. Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1. The demonstration that DNA polymerase II is a component of the SOS response in E. coli suggests that it plays an important role in DNA repair and/or mutagenesis.Of the three distinct DNA polymerizing activities purified from the bacterium Escherichia coli, the role of DNA polymerase II (pol II) is the least understood. The function of DNA polymerase I in replication and repair has been well documented (1), and the DNA polymerase III holoenzyme constitutes the replicative polymerase in this organism (2). Remarkably, however, the biological role of pol II has not been determined, and no phenotype has been identified for mutants (polB) deficient in this activity (3, 4).Recently, we reported that purified pol II catalyzed the insertion ofnucleotides opposite defined abasic sites in model templates (5). This insertion and the subsequent extension steps are thought to be critical features of "lesion bypass," which likely accounts for targeted mutagenesis in prokaryotes (6, 7). That pol II could incorporate a nucleotide (preferably dAMP) opposite a noncoding site in DNA was consistent with a role for this activity in mutagenesis. Of equal significance was our observation (5) that the levels of pol II increased in cells exposed to agents that block replication (nalidixate) and that this apparent increase in pol II activity was regulated by the lexA gene, which controls expression of the SOS response in E. coli (8). Induced mutagenesis in bacteria requires induction of components of the SOS regu-

In order to examine the possible role of Escherichia coli DNA polymerase H in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded 4X174 transfecting DNA containing individual lesions. No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase H was observed. Furthermore, DNA polymerase H did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint. Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II.The cellular function of DNA polymerase II (Pol II) of Escherichia coli has eluded biochemists and geneticists since it was originally identified over 20 years ago (20,21,28 (la, 19). Taken together, these data suggest that DNA Pol II is involved in cellular DNA repair.It has been commonly thought and most data have suggested that the DNA polymerase involved in lesion bypass and mutagenesis is the DNA Pol III holoenzyme (4, 5, 14), and genetic studies have shown that RecA, UmuC, and UmuD are required for mutagenesis produced by UV lesions (33) and abasic sites (26). Weigle reactivation (35), an increase in survival of damaged phage or phage DNA, also requires these gene products (6,11,12 DNA containing DNA damage. Weigle reactivation of singlestranded and duplex DNA phages and transfecting DNA has been used as a measure of lesion bypass in a number of systems. It is a sensitive and simple method, since increases or decreases in survival are quite easily scored. We elected to examine the potential role of Pol II in the survival of two lesions, thymine glycol and the cyclobutane pyrimidine dimer, that are strong blocks to DNA polymerases in uninduced cells but are efficiently bypassed in SOS-induced cells. Thymine glycol, an oxidative DNA damage that is lethal in uninduced cells, is the most efficiently bypassed lesion examined to date in induced cells (34). A total of 60 to 70% of thymine glycols are bypassed in both single-stranded (16) and duplex (25) DNAs, whereas 30 to 40% of cyclobutane pyrimidine dimers are bypassed (38). For both lesions, we used single-stranded damage-containing DNA to remove any possible effects of excision and recombination repair processing from the endpoint. Figure 1 shows the survival response of damaged DNA containing either thymine glycols (Fig. 1A) or cyclobutane pyrimidine dimers (Fig. 1B) with increasing damage-inducing UV light dose to the host. As can be seen in both cases, Weigle reactivation was substantial, but no difference was observed whether the strain contained or lacked DNA PolII. Furthermore, the results shown here for the dinA Mud(Apr lac) fusion were also obtained with a polB point mutant, a polB deletion (SH2101; unpublished observations), or a double mutant defective in both DNA PolI and Pol II (poLA polB; data not shown). Thus, DNA Pol II does not appear to be involved in Weigle reactivation of readily bypassable lesions.We and o...

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