Requirement of RAD52 group genes for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, replication through DNA lesions is promoted by Rad6-Rad18-dependent processes that include translesion synthesis by DNA polymerases eta and zeta and a Rad5-Mms2-Ubc13-controlled postreplicational repair (PRR) pathway which repairs the discontinuities in the newly synthesized DNA that form opposite from DNA lesions on the template strand. Here, we examine the contributions of the RAD51, RAD52, and RAD54 genes and of the RAD50 and XRS2 genes to the PRR of UV-damaged DNA. We find that deletions of the RAD51, RAD52, and RAD54 genes impair the efficiency of PRR and that almost all of the PRR is inhibited in the absence of both Rad5 and Rad52. We suggest a role for the Rad5 pathway when the lesion is located on the leading strand template and for the Rad52 pathway when the lesion is located on the lagging strand template. We surmise that both of these pathways operate in a nonrecombinational manner, Rad5 by mediating replication fork regression and template switching via its DNA helicase activity and Rad52 via a synthesis-dependent strand annealing mode. In addition, our results suggest a role for the Rad50 and Xrs2 proteins and thereby for the MRX complex in promoting PRR via both the Rad5 and Rad52 pathways.     

DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.     

RAD29 and RAD31--new genes from Saccharomyces cerevisiae yeasts, participating in control of DNA repair. Isolation and genetic study of mutants

Base excision repair (BER) and nucleotide excision repair (NER) are two main cellular responses to DNA damage induced by various physical and chemical factors. After exposure of the strain that carries the NER-blocking rad2 mutation to UV light, several mutants hypersensitive to the UV light lethal action and simultaneously sensitive to methylmethanesulphonate (MMS) were isolated. Two of these mutants (Uvs64 and Uvs212) were examined in detail. The mutants were found to carry recessive, monogenically inherited lesions that had pleiotropic, though different, phenotypes: both mutants were also sensitive to nitrous acid (HNO2), whereas Uvs212 was sensitive to hydrogen peroxide as well. Moreover, the homozygote for the uvs212 mutation, but not for uvs64, blocks the sporulation. Since the mutations examined were not allelic to any of the known rad mutations that cause MMS sensitivity or to each other, it is concluded that two new genes involved in the control of yeast DNA repair were detected. Furthermore, these genes were mapped to different regions of the right arm of chromosome 2 where repair genes were not found. Thus, two new genes, designated RAD29(UVS64) and RAD31(UVS212) and probably involved in base excision repair, were identified.     

The structure and function of RAD6 and RAD18 DNA repair genes of Saccharomyces cerevisiae

The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplication repair of discontinuities occurring in newly synthesized DNA following exposure to uv light. In addition, rad6 mutants are highly defective in mutagenesis induced by uv and other DNA damaging agents and in sporulation. RAD6 encodes a protein of 172 amino acids with a highly acidic carboxyl terminus. Deletion of the carboxyl terminal 23 residues, 20 of which are acidic, has little or no effect on uv sensitivity or uv mutagenesis, but sporulation is greatly reduced. Addition of the first four residues of the polyacidic tail restores sporulation to 50% the level observed in RAD+/RAD+ diploids. RAD6 protein has been previously shown to be a ubiquitin-conjugating (E2) enzyme that attaches ubiquitin to histones H2A and H2B in vitro. Our experiments show that deletion of varying lengths of the polyacidic tail of RAD6 protein greatly reduces its ubiquitin-conjugating activity. The RAD18 encoded protein contains features which suggest that it binds DNA and nucleotides. Ten of the 12 cysteine residues occur in regions that could form zinc finger domains for nucleic acid binding. The other interesting feature in RAD18 protein is the presence of a putative nucleotide binding sequence. The possible in vivo functions of the RAD6 and RAD18 proteins are discussed.     

LIG4 and RAD52 DNA repair genes polymorphisms and systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with a strong genetic background. Nevertheless, SLE might also be triggered due to environmental factors, such as UV light exposure. DNA double strand breaks (DSBs) may be induced secondarily by UV radiation, increasing DNA immunogenicity and in SLE patients DNA repair is diminished, allowing the accumulation of DSBs and genomic instability. LIG4 and RAD52 genes play important roles in DNA repair mechanisms and a recent microarray analysis showed their differential expression in active SLE patients. In this study we investigated a potential association between LIG4 and RAD52 single nucleotide polymorphisms (SNPs) and SLE predisposition in a Southeast Brazilian population. We assessed four Tag SNPs in LIG4 and three in RAD52 gene region, encompassing most of the gene sequence, in 158 SLE patients and 212 healthy controls. We also performed SNPs analysis considering clinical manifestation, gender and ethnicity in SLE patients. Our data did not show association between LIG4 and RAD52 SNPs and SLE, its clinical manifestations or ethnicity in the tested population. The analysis regarding ethnicity and SLE clinical manifestations indicated Caucasian-derived patients as more susceptible to cutaneous and hematological alterations than the African-derived. To our knowledge, this is the first association study involving LIG4 and RAD52 genes and SLE predisposition.     

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.     

The Saccharomyces cerevisiae RAD9, RAD17 and RAD24 genes are required for suppression of mutagenic post-replicative repair during chronic DNA damage

In Saccharomyces cerevisiae, a DNA damage checkpoint in the S-phase is responsible for delaying DNA replication in response to genotoxic stress. This pathway is partially regulated by the checkpoint proteins Rad9, Rad17 and Rad24. Here, we describe a novel hypermutable phenotype for rad9Δ, rad17Δ and rad24Δ cells in response to a chronic 0.01% dose of the DNA alkylating agent MMS. We report that this hypermutability results from DNA damage introduction during the S-phase and is dependent on a functional translesion synthesis pathway. In addition, we performed a genetic screen for interactions with rad9Δ that confer sensitivity to 0.01% MMS. We report and quantify 25 genetic interactions with rad9Δ, many of which involve the post-replication repair machinery. From these data, we conclude that defects in S-phase checkpoint regulation lead to increased reliance on mutagenic translesion synthesis, and we describe a novel role for members of the S-phase DNA damage checkpoint in suppressing mutagenic post-replicative repair in response to sublethal MMS treatment.     

DNA damage and repair in gastric cancer--a correlation with the hOGG1 and RAD51 genes polymorphisms

Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by ³²P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N⁶-yl]-aristolactam I, 7-[deoxyadenosin-N⁶-yl]-aristolactam II and 7-[deoxyguanosin-N²-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10⁸ nucleotides in liver and 95–4598 adducts/10⁸ nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 × 10⁻⁶ in liver compared with the MFs of 78–1319 × 10⁻⁶ that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.     

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.?cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.     

Role of mismatch repair in the fidelity of RAD51- and RAD59-dependent recombination in Saccharomyces cerevisiae

To prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway. To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae. The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91 and 100% identical sequences demonstrated differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59. In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displayed distinct patterns of inhibition by different members of the MMR pathway. Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination were similar in rad51 strains, the loss of MSH2 caused a greater loss in homeologous recombination suppression than did the loss of MLH1 in a rad59 strain. The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells.     

Genetic evidence for ongoing balanced selection at human DNA repair genes ERCC8, FANCC, and RAD51C

Using the 2.6 million single nucleotide polymorphism (SNP) genotype datasets from Perlegen Sciences and the Haplotype Map (HapMap) project (Phase I freeze), a probabilistic search for the landscape exhibited by positive Darwinian selection was conducted (Wang et al., 2006). By sorting each high frequency allele by homozygosity, we search for the expected decay of adjacent SNP linkage disequilibrium (LD) at recently selected alleles, eliminating the need for inferring haplotype. We designate this approach the LD decay (LDD) test. Cluster analysis indicates that approximately 3000 sites of recent inferred selection are present in human DNA, representing approximately 1800 genes. Prior simulation studies (Wang et al., 2006) indicate that this novel LDD test, at the Mb scale employed, effectively distinguishes selection from other causes of extensive LD, such as inversions, population bottlenecks and admixture. Based on over-representation analysis, these prior studies have shown that several predominant biological themes are common in inferred selected alleles, including genes involved with DNA metabolism and repair. Here, we show that three of these DNA repair genes, ERCC8, Fanconi Anemia Complementation Group C (FANCC), and RAD51C, exhibit genomic architectures consistent with ongoing balanced selection over the last 40,000-50,000 years.     

Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae.

We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.     

RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis

Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability.