Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Genetic analysis of homologous DNA recombination in vertebrate somatic cells

The maintenance of genomic stability and the ability to repair induced DNA damage in vertebrate cells require homologues of the yeast RAD52 epistasis group genes. The homologous recombination carried out by the products of these genes is essential and appears to be closely linked to DNA replication. Defects in recombination and associated activities are implicated in human cancer. This review summarises recent biochemical and genetic findings on the roles played by the vertebrate RAD52 group gene products in recombination. We describe the phenotypic analysis of genetically engineered mammalian and chicken mutants of homologous recombination genes.     

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.     

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.     

Cells expressing murine RAD52 splice variants favor sister chromatid repair

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.     

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.     

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.     

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.     

Epistasis analysis between homologous recombination genes in Saccharomyces cerevisiae identifies multiple repair pathways for Sgs1, Mus81-Mms4 and RNase H2

The DNA repair genes SGS1 and MUS81 of Saccharomyces cerevisiae are thought to control alternative pathways for the repair of toxic recombination intermediates based on the fact that sgs1Δ mus81Δ synthetic lethality is suppressed in the absence of homologous recombination (HR). Although these genes appear to functionally overlap in yeast and other model systems, the specific pathways controlled by SGS1 and MUS81 are poorly defined. Epistasis analyses based on DNA damage sensitivity previously indicated that SGS1 functioned primarily downstream of RAD51, and that MUS81 was independent of RAD51. To further define these genetic pathways, we carried out a systematic epistasis analysis between the RAD52-epistasis group genes and SGS1, MUS81, and RNH202, which encodes a subunit of RNase H2. Based on synthetic-fitness interactions and DNA damage sensitivities, we find that RAD52 is epistatic to MUS81 but not SGS1. In contrast, RAD54, RAD55 and RAD57 are epistatic to SGS1, MUS81 and RNH202. As expected, SHU2 is epistatic to SGS1, while both SHU1 and SHU2 are epistatic to MUS81. Importantly, loss of any RNase H2 subunit on its own resulted in increased recombination using a simple marker-excision assay. RNase H2 is thus needed to maintain genome stability consistent with the sgs1Δ rnh202Δ synthetic fitness defect. We conclude that SGS1 and MUS81 act in parallel pathways downstream of RAD51 and RAD52, respectively. The data further indicate these pathways share common components and display complex interactions.     

The role of homologous recombination processes in the repair of severe forms of DNA damage in mammalian cells

The role of homologous recombination processes in the repair of severe forms of DNA damage is reviewed, with particular attention to the functions of members of the recA/RAD51 family of genes. In the yeast Saccharomyces cerevisiae, several of the gene products involved in homologous recombination repair (HRR) have been studied in detail, and a picture is beginning to emerge of the repair mechanism for DNA double-strand breaks. Knowledge is fragmentary for other eukaryotic organisms and for other types of DNA damage. In mammalian cells, while it has been known for some years that HRR occurs, the relative importance of the process in repairing DNA damage is unknown and very few of the gene products involved have been identified. Very recently, a number of RAD51-like genes have been identified in mammals, either through cloning genes complementing cell lines sensitive to DNA-damaging agents (XRCC2, XRCC3), or through homology searches (RAD51L1, RAD51L2, RAD51L3). As yet the role of these genes and their possible functions are speculative, although the combination of sequence conservation and gene expression patterns suggest that they function in HRR pathways.     

RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates

DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system. RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.     

Dds20 operates in cds1-independent mechanism of tolerance to UV-induced DNA damage in Schizosaccharomyces pombe cells

Repair of DNA double-stranded breaks caused by ionizing radiation or cellular metabolization, homologous recombination, is an evolutionary conserved process controlled by RAD52 group genes. Genes of recombinational repair also play a leading role in the response to DNA damage caused by UV light. Cells with deletion in gene dds20 of recombinational repair were shown to manifest hypersensitivity to the action of UV light at lowered incubation temperature. Epistatic analysis revealed that dds20+ is not a member of the NER and UVER gene groups responsible for the repair of DNA damage induced by UV light. The Dds protein has functions in the Cds1-independent mechanism of UV damage tolerance of DNA.     

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G2 checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-D (null) mutant is defective in both S and G2 checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G2 checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1, and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G2 checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G2-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G2 arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.     

Requirement of Mismatch Repair Genes Msh2 and Msh3 in the Rad1-Rad10 Pathway of Mitotic Recombination in Saccharomyces Cerevisiae

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3Δ mutation has an effect on recombination similar to that of the rad1Δ and rad10Δ mutations. The msh2Δ mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.     

Effects of mutations in DNA repair genes on formation of ribosomal DNA circles and life span in Saccharomyces cerevisiae

A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.     

The effects of RAD52 epistasis group genes on various types of spontaneous mitotic recombination in Saccharomyces cerevisiae

The role of RAD52 epistasis group genes on spontaneous mitotic recombination was examined using three different types of spontaneous mitotic recombination in Saccharomyces cerevisiae. The spontaneous recombination between homologous sequences in a plasmid and a chromosome was essentially unaffected by null mutations in any of the RAD52 epistasis group genes. Recombination between genes in separate autonomously replicating plasmids was reduced 833-fold in a rad52 null mutant, but only 2- to at most 20-fold in rad50, 51, 54, 55, 57 null mutants. Recombination between tandemly repeated heteroalleles in an autonomously replicating plasmid was reduced almost 100-fold in a rad52 null mutant, but is either unaffected or slightly increased in rad50, 51, 54, 55, 57 null mutants. The finding that RAD50, 51, 54, 55, 57 are dispensable or marginally involved in these spontaneous recombinations suggests further that spontaneous mitotic recombination in S. cerevisiae might be processed by other than RAD52 epistasis group.     

Over-expression of RAD51 or RAD54 but not RAD51/4 enhances extra-chromosomal homologous recombination in the human sarcoma (HT-1080) cell line

RAD51 and RAD54, members of the RAD52 epistasis group, play key roles in homologous recombination (HR). The efficiency of homologous recombination (HR) can be increased by over-expression of either of them. A vector that allows co-expression of RAD51 and RAD54 was constructed to investigate interactions between the two proteins during extra-chromosomal HR. The efficiency of extra-chromosomal HR evaluated by GFP extra-chromosomal HR was enhanced (110-245%) in different transfected Human sarcoma (HT-1080) cell colonies. We observed that RAD51 clearly promotes extra-chromosomal HR; however, the actions of RAD54 in extra-chromosomal HR were weak. Our data suggest that RAD51 may function as a universal factor during HR, whereas RAD54 mainly functions in other types of HR (gene targeting or intra-chromosomal HR), which involves interaction with chromosomal DNA.