Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination

An intrachromosomal recombination assay that monitors events between alleles of the ade2 gene oriented as inverted repeats was developed. Recombination to adenine prototrophy occurred at a rate of 9.3 x 10(-5)/cell/generation. Of the total recombinants, 50% occurred by gene conversion without crossing over, 35% by crossover and 15% by crossover associated with conversion. The rate of recombination was reduced 3,000-fold in a rad52 mutant, but the distribution of residual recombination events remained similar to that seen in the wild type strain. In rad51 mutants the rate of recombination was reduced only 4-fold. In this case, gene conversion events unassociated with a crossover were reduced 18-fold, whereas crossover events were reduced only 2.5-fold. A rad51 rad52 double mutant strain showed the same reduction in the rate of recombination as the rad52 mutant, but the distribution of events resembled that seen in rad51. From these observations it is concluded that (i) RAD52 is required for high levels of both gene conversions and reciprocal crossovers, (ii) that RAD51 is not required for intrachromosomal crossovers, and (iii) that RAD51 and RAD52 have different functions, or that RAD52 has functions in addition to those of the Rad51/Rad52 protein complex.     

Gene conversion in the chicken immunoglobulin locus: a paradigm of homologous recombination in higher eukaryotes

Gene conversion was first defined in yeast as a type of homologous recombination in which the donor sequence does not change. In chicken B cells, gene conversion builds the antigen receptor repertoire by introducing sequence diversity into the immunoglobulin genes. Immunoglobulin gene conversion continues at high frequency in an avian leukosis virus induced chicken B cell line. This cell line can be modified by homologous integration of transfected DNA constructs offering a model system for studying gene conversion in higher eukaryotes. In search for genes which might participate in chicken immunoglobulin gene conversion, we have identified chicken counterparts of the yeast RAD51, RAD52, and RAD54 genes. Disruption and overexpression of these genes in the chicken B cell line may clarify their role in gene conversion and gene targeting.     

Stimulation by Rad52 of yeast Rad51-mediated recombination

In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homologue of the recA gene and the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologues of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Here we show that Rad52 protein stimulates Rad51 reactions and that binding to Rad51 is necessary for this stimulatory effect. We conclude that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments.     

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.     

Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA

RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.     

The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast

The rad52-1 mutation prevents homothallic mating type interconversion and reduces mitotic recombination in yeast. It has been previously reported that rad52-1 abolishes meiotic recombination. These data suggest either that a generalized recombination function(s) is required for mating type switching or that generalized recombination and specific homothallic functions are jointly controlled by the RAD52 gene. The rad52-1 mutation affects the interconversion of the two yeast mating types (a and alpha) differently, suggesting that the interconversion process is not equivalent for both mating types. This type of asymmetry is not predicted by current models of homothallic switching.     

Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.     

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.     

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.     

Analysis of spontaneous and double-strand break-induced recombination in rad mutants of S. pombe

Schizosaccharomyces pombe strains containing direct repeats of adeó heteroalleles separated by a functional uro4+ gene, and a DNA site for induction of a double-strand break (DSB), have been used to analyze pathways of spontaneous and DSB-induced intrachromosomal mitotic recombination. These substrates yield Ade+ Ura+ convertants or Ade+ Ura- deletions, by the DSB/gap repair and single-strand annealing (SSA) pathways of recombination, respectively. In S. cerevisiae, the DSB/gap repair pathway is RAD52 dependent, and the RAD1 and RAD10 genes are involved in the SSA pathway. We have sought to understand the genetic control of the pathways of mitotic recombination in S. pombe by determining the effects of mutations in six rad genes involved in DNA repair: rad1 and rad3 involved in checkpoint control in response to unreplicated or damaged DNA; rad5 (homologue of S. cerevisiae RAD3) and rad10 (homologue of S. cerevisiae RAD1) involved in nucleotide excision repair; rad21 and rad22 (homologue of S. cerevisiae RAD52) involved in the repair of ionizing radiation-induced DNA damage. The results suggest that the genetic control of the pathways of spontaneous and DSB-induced mitotic intrachromosomal recombination in S. pombe is different from that in S. cerevisiae.     

Homologous recombination is required for the viability of rad27 mutants

The RAD27/RTH1 gene of Saccharomyces cerevisiae encodes a structural and functional homolog of the 5'-3' exonuclease function of Escherichia coli DNA polymerase I. Four alleles of RAD27 were recovered in a screen for hyper-recombination, a phenotype also displayed by polA mutants of E.coli. All four rad27 mutants showed similar high levels of mitotic recombination, but varied in their growth rate at various temperatures, and sensitivity to the DNA damaging agent methyl methane sulfonate. Dependence of viability of rad27 strains on recombination was determined by crossing a strain containing a null allele of RAD27 to strains containing a mutation in either the RAD1, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11, XRS2 or RAD59 gene. In no case were viable spore products recovered that contained both mutations. Elimination of the non-homologous end-joining pathway did not affect the viability of a rad27 strain. This suggests that lesions generated in the absence of RAD27 must be processed by homologous recombination.     

NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae

Distinct classes of sporulation-specific genes are sequentially expressed during the process of spore formation in Saccharomyces cerevisiae. The transition from expression of early meiotic genes to expression of middle sporulation-specific genes occurs at about the time that cells exit from pachytene and form the meiosis I spindle. To identify genes encoding potential regulators of middle sporulation-specific gene expression, we screened for mutants that expressed early meiotic genes but failed to express middle sporulation-specific genes. We identified mutant alleles of RPD3, SIN3, and NDT80 in this screen. Rpd3p, a histone deacetylase, and Sin3p are global modulators of gene expression. Ndt80p promotes entry into the meiotic divisions. We found that entry into the meiotic divisions was not required for activation of middle sporulation genes; these genes were efficiently expressed in a clb1 clb3 clb4 strain, which fails to enter the meiotic divisions due to reduced Clb-dependent activation of Cdc28p kinase. In contrast, middle sporulation genes were not expressed in a dmc1 strain, which fails to enter the meiotic divisions because a defect in meiotic recombination leads to a RAD17-dependent checkpoint arrest. Expression of middle sporulation genes, as well as entry into the meiotic divisions, was restored to a dmc1 strain by mutation of RAD17. Our studies also revealed that NDT80 was a temporally distinct, pre-middle sporulation gene and that its expression was reduced, but not abolished, on mutation of DMC1, RPD3, SIN3, or NDT80 itself. In summary, our data indicate that Ndt80p is required for expression of middle sporulation genes and that the activity of Ndt80p is controlled by the meiotic recombination checkpoint. Thus, middle genes are expressed only on completion of meiotic recombination and subsequent generation of an active form of Ndt80p.     

The Saccharomyces cerevisiae RAD54 gene is important but not essential for natural homothallic mating-type switching

Homothallic Saccharomyces cerevisiae strains switch their mating-type in a specific gene conversion event induced by a DNA double strand break made by the HO endonuclease. The RAD52 group genes control recombinational repair of DNA double strand breaks, and we examined their role in native homothallic mating-type switching. Surprisingly, we found that the Rad54 protein was important but not essential for mating-type switching under natural conditions. As an upper limit, we estimate that 29% of the rad54 spore clones can successfully switch their mating-type. The RAD55 and RAD57 gene products were even less important, but their presence increased the efficiency of the process. In contrast, the RAD51 and RAD52 genes are essential for homothallic mating-type switching. We propose that mating-type switching in RAD54 mutants occurs stochastically with a low probability, possibly reflecting different states of chromosomal structure.     

The Saccharomyces cerevisiae RAD9 checkpoint reduces the DNA damage-associated stimulation of directed translocations

Genetic instability in the Saccharomyces cerevisiae rad9 mutant correlates with failure to arrest the cell cycle in response to DNA damage. We quantitated the DNA damage-associated stimulation of directed translocations in RAD9+ and rad9 mutants. Directed translocations were generated by selecting for His+ prototrophs that result from homologous, mitotic recombination between two truncated his3 genes, GAL1::his3-delta5' and trp1::his3-delta3'::HOcs. Compared to RAD9+ strains, the rad9 mutant exhibits a 5-fold higher rate of spontaneous, mitotic recombination and a greater than 10-fold increase in the number of UV- and X-ray-stimulated His+ recombinants that contain translocations. The higher level of recombination in rad9 mutants correlated with the appearance of nonreciprocal translocations and additional karyotypic changes, indicating that genomic instability also occurred among non-his3 sequences. Both enhanced spontaneous recombination and DNA damage-associated recombination are dependent on RAD1, a gene involved in DNA excision repair. The hyperrecombinational phenotype of the rad9 mutant was correlated with a deficiency in cell cycle arrest at the G2-M checkpoint by demonstrating that if rad9 mutants were arrested in G2 before irradiation, the numbers both of UV- and gamma-ray-stimulated recombinants were reduced. The importance of G2 arrest in DNA damage-induced sister chromatid exchange (SCE) was evident by a 10-fold reduction in HO endonuclease-induced SCE and no detectable X-ray stimulation of SCE in a rad9 mutant. We suggest that one mechanism by which the RAD9-mediated G2-M checkpoint may reduce the frequency of DNA damage-induced translocations is by channeling the repair of double-strand breaks into SCE.     

Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations

To directly determine whether recombinational repair of double-strand breaks (DSBs) can occur between heterologous chromosomes and lead to chromosomal rearrangements in mammalian cells, we employed an ES cell system to analyze recombination between repeats on heterologous chromosomes. We found that recombination is induced at least 1000-fold following the introduction of a DSB in one repeat. Most (98%) recombinants repaired the DSB by gene conversion in which a small amount of sequence information was transferred from the unbroken chromosome onto the broken chromosome. The remaining recombinants transferred a larger amount of information, but still no chromosomal aberrations were apparent. Thus, mammalian cells are capable of searching genome-wide for sequences that are suitable for DSB repair. The lack of crossover events that would have led to translocations supports a model in which recombination is coupled to replication.     

Gene conversions and crossing over during homologous and homeologous ectopic recombination in Saccharomyces cerevisiae

The pma1-105 mutation reduces the activity of the yeast plasma membrane H(+)-ATPase and causes cells to be both low pH and ammonium ion sensitive and resistant to the antibiotic hygromycin B. Revertants that can grow at pH 3.0 and on ammonium-containing plates frequently arise by ectopic recombination between pma1-105 and PMA2, a diverged gene that shares 85% DNA sequence identity with PMA1. The gene conversion tracts of revertants of pma1-105 were determined by DNA sequencing the hybrid PMA1::PMA2 genes. Gene conversion tracts ranged from 18-774 bp. The boundaries of these replacements were short (3-26 bp) regions of sequences that were identical between PMA1 and PMA2. These boundaries were not located at the regions of greatest shared identity between the two PMA genes. Similar results were obtained among low pH-resistant revertants of another mutation, pma1-147. One gene conversion was obtained in which the resulting PMA1::PMA2 hybrid was low pH-resistant but still hygromycin B-resistant. This partially active gene differs from a wild-type revertant only by the presence of two PMA2-encoded amino acid substitutions. Thus, some regions of PMA2 are not fully interchangeable with PMA1. We have also compared the efficiency of recombination between pma1-105 and either homeologous PMA2 sequence or homologous PMA1 donor sequences inserted at the same location. PMA2 x pma1-105 recombination occurred at a rate approximately 75-fold less than PMA1 x pma1-105 events. The difference in homology between the interacting sequences did not affect the proportion of gene conversion events associated with a cross-over, as in both cases approximately 5% of the Pma+ recombinants had undergone reciprocal translocations between the two chromosomes carrying pma1-105 and the donor PMA sequences. Reciprocal translocations were identified by a simple and generally useful nutritional test.