Inactivation of RAD52 aggravates RAD54 defects in mice but not in Schizosaccharomyces pombe

RAD52 and RAD54 genes from Saccharomyces cerevisiae are required for double-strand break repair through homologous recombination and show epistatic interactions i.e., single and double mutant strains are equally sensitive to DNA damaging agents. In here we combined mutations in RAD52 and RAD54 homologs in Schizosaccharomyces pombe and mice. The analysis of mutant strains in S. pombe demonstrated nearly identical sensitivities of rhp54, rad22A and rad22B double and triple mutants to X-rays, cis-diamminedichloroplatinum and hydroxyurea. In this respect, the fission yeast homologs of RAD54 and RAD52 closely resemble their counterparts in S. cerevisiae. To verify if inactivation of RAD52 affects the DNA damage sensitivities of RAD54 deficient mice, several endpoints were studied in double mutant mice and in bone marrow cells derived from these animals. Haemopoietic depression in bone marrow and the formation of micronuclei after in vivo exposure to mitomycine C (MMC) was not increased in either single or double mutant mice in comparison to wildtype animals. The induction of sister chromatid exchanges in splenocytes was slightly reduced in the RAD54 mutant. A similar reduction was detected in the double mutant. However, a deficiency of RAD52 exacerbates the MMC survival of RAD54 mutant mice and also has a distinct effect on the survival of bone marrow cells after exposure to ionizing radiation. These findings may be explained by additive defects in HR in the double mutant but may also indicate a more prominent role for single-strand annealing in the absence of Rad54.     

A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.     

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.     

Multiple recombination pathways for sister chromatid exchange in Saccharomyces cerevisiae: role of RAD1 and the RAD52 epistasis group genes

Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching. To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires specific genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-Δ5′ and his3-Δ3′::HOcs. SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-Δ3′::HOcs. Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damage-associated SCE but not for spontaneous SCE. RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs. In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants. We propose that gap repair mechanisms are important in DNA damage-associated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE.     

Inducible error-prone repair in yeast. Suppression by heat shock

The production of reversion mutations in wild-type, diploid Saccharomyces cerevisiae by the alkylating agents N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU) was suppressed in cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same cells previously treated with a heat shock, or the protein synthesis inhibitor, cycloheximide. The same treatment after mutagen exposure did not lower the induced mutation frequency. In split-dose experiments, a first MNNG exposure prevented subsequent heat (or cycloheximide) treatment from blocking mutation by a second, later mutagen exposure. These data suggest that, in yeast, MNNG or MNU induces an error-prone DNA-repair system, and that this induction is blocked by protein-synthesis inhibitors. The specificity of this system for different types of DNA damage was investigated using a variety of other mutagenic agents. A prior heat shock did not suppress mutation produced by exposure to ethyl methanesulfonate, ethylnitrosourea, 8-methoxypsoralen + UVA, or gamma-radiation. Partial suppression was observed in cells exposed to methyl methanesulfonate or to 254-nm ultraviolet light. These results indicate that, unlike the SOS system of E. coli, this inducible error-prone process of yeast is responsive to only certain mutagens. Heat shock suppression of mutation produced by MNNG exposure was also demonstrated in wild-type haploid cells, as well as haploid strains mutant in representative genes of the RAD52 epistasis group (rad52, rad53, rad54), the RAD3 epistasis group (rad1, rad2, rad3) and the RAD6 epistasis group (rad9, rad18). The rad6 mutant itself was immutable with MNNG and therefore untestable by these techniques. These data indicate that this error-prone repair system is not absolutely dependent on the integrity of the RAD52 (recombination) or the RAD3 (excision) systems, or on at least some parts of the RAD6 system.     

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.     

Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae

The SGS1 gene of Saccharomyces cerevisiae is homologous to the genes that are mutated in Bloom's syndrome and Werner's syndrome in humans. Disruption of SGS1 results in high sensitivity to methyl methanesulfonate (MMS), poor sporulation, and a hyper-recombination phenotype including recombination between heteroalleles. In this study, we found that SGS1 forms part of the RAD52 epistasis group when cells are exposed to MMS. Exposure to DNA-damaging agents causes a striking, Rad52-dependent, increase in heteroallelic recombination in wild-type cells, but not in sgs1 disruptants. However, in the absence of DNA damage, the frequency of heteroallelic recombination in sgs1 disruptants was several-fold higher than in wild-type cells, as described previously. These results imply a function for Sgs1: it acts to suppress spontaneous heteroallelic recombination, and to promote DNA damage-induced heteroallelic recombination.     

Functional conservation of the yeast and Arabidopsis RAD54-like genes

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.     

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.     

Expression of the RAD1 and RAD3 genes of Saccharomyces cerevisiae is not affected by DNA damage or during the cell division cycle

Summary The RAD1 and RAD3 genes of Saccharomyces cerevisiae are required for excision repair of UV damaged DNA. In addition, the RAD3 gene is essential since rad3 deletions are recessive lethals. We have examined the induction of the RAD1 and RAD3 genes by DNA damage and during the cell division cycle. We have made fusions of the RAD1 and RAD3 genes with the Escherichia coli lacZ gene encoding -galactosidase. -galactosidase activity was measured in a Rad⁺ yeast strain containing the RAD1-lacZ or the RAD3-lacZ fusion, either in a multicopy replicating plasmid or as a single copy integrant resulting from transformation with an integrating plasmid which transforms yeast by homologous recombination in the yeast genome. No induction of -galactosidase activity occurred after ultraviolet light (UV) or 4-nitroquinoline-1-oxide (NQO) treatment. Haploid cells of mating type a were synchronized by treatment with factor and -galactosidase activity was determined during different cell cycle stages. No change in -galactosidase activity was observed in the strain containing the RAD1-lacZ or the RAD3-lacZ fusion integrated in the yeast genome.     

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable,ultraviolet-induced DNA damage.

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.