A reexamination of the role of the RAD52 gene in spontaneous mitotic recombination

Summary The RAD52 gene is required for much of the recombination that occurs in Saccharomyces cerevisiae. One of the two commonly utilized mutant alleles, rad52-2, increases rather than reduces mitotic recombination, yet in other respects appears to be a typical rad52 mutant allele. This raises the question as to whether RAD52 is really necessary for mitotic recombination. Analysis of a deletion/insertion allele created in vitro indicates that the null mutant phenotype is indeed a deficiency in mitotic recombination, especially in gene conversion. The data also indicate that RAD52 is required for crossing-over between at least some chromosomes. Finally, examination of the behavior of a replicating plasmid in rad52-1 strains indicates that the frequency of plasmid integration is substantially reduced from that in wild type, a conclusion consistent with a role for RAD52 in reciprocal crossing-over. Analysis of recombinants arising in rad52-2 strains suggests that this allele may result in the increased activity of a RAD52-independent recombinational pathway.     

The RAD50 gene,a member of the double strand break repair epistasis group,is not required for spontaneous mitotic recombination in yeast

Summary Mutations in the RAD50 gene of Saccharomyces cerevisiae have been shown to reduce double strand break repair, meiotic recombination, and radiation-inducible mitotic recombination. Several different point mutations (including ochre and amber alleles) have been previously examined for effects on spontaneous mitotic recombination and did not reduce the frequency of recombination. Instead, the rad50 mutations conferred a moderate hyper-rec phenotype. This paper examines a deletion/interruption allele of RAD50 that removes 998 of 1312 amino acids and adds 1.1 kb of foreign DNA. The results clearly indicate that spontaneous mitotic recombination can occur in the absence of RAD50; in fact, the frequency of recombination is elevated over the wild-type cell. One possible interpretation of these observations is that the initiating lesion in spontaneous recombination events in mitosis might not be a double strand break.     

Interactions among mutations affecting spontaneous mutation,mitotic recombination,and DNA repair in yeast

The mutant alleles mms9-1, mms13-1, or mms21-1 of Saccharomyces cerevisiae confer pleiotropic effects, including sensitivity to the alkylating agent methyl methanesulfonate, elevations in spontaneous mutation and mitotic recombination, defects in meiosis, and cross-sensitivity to radiation. We constructed double-mutant strains containing an mms mutation and a defect in either excision repair, mutagenic repair, or recombinational repair and measured the levels of spontaneous mutation and mitotic reombination. Double mutants lacking excision repair show elevations in spontaneous mutation but with predominantly unchanged levels of mitotic recombination. RAD52 function was required for the expression of the hyper-recombination phenotype of the mms9-1, mms13-1, and mms21-1 alleles; double mutants displayed the very low recombination levels characteristic of rad52 mutants. Phenotypes of double mutants containing one of the mms alleles and either of the hyper-recombination/mutator rad6-1 or rad3-102 alleles suggest that the mutagenic lesions in mms strains may not be identical to the recombinogenic lesions.     

Cloning and characterization of the yeast RAD1 homolog gene (mus-38) from Neurospora crassa: evidence for involvement in nucleotide excision repair

A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR. The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32–37% identity to the XPF/ERCC4 protein family. The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene. Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38. Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant. The double mutant also was synergistically more sensitive to UV than either single mutant. The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene. Received: 27 November 1997 / 28 January 1998     

RPD3 (REC3) mutations affect mitotic recombination in Saccharomyces cerevisiae

Prior research identified the recessive rec3-1ts mutation in Saccharomyces cerevisiae which, in homozygous diploid cells, confers a conditional phenotype resulting in reduced levels of spontaneous mitotic recombination and loss of sporulation at the restrictive temperature of 36 °C. We found that a 3.4-kb genomic fragment that complements the rec3-1ts/rec3-1ts mutation and which maps to chromosome XIV, is identical to RPD3, a gene encoding a histone de-acetylase. Sporulation is reduced in homozygous diploid strains containing the rec3-1ts allele at 24 °C, suggesting that this allele of RPD3 encodes a gene product with a reduced function. Sporulation is abolished in diploid strains homozygous for the rpd3Δ or rec3-1ts alleles, as well as in rpd3Δ/rec3-1ts heteroallelic diploids, at the non-permissive temperature. Acid-phosphatase expression has been shown to be RPD3 dependent. We found that acid-phosphatase activity is greater in diploid strains homozygous for the temperature-sensitive rec3-1ts allele than in RPD3/RPD3 strains and increased further when mutant strains are grown at 36 °C. We also tested the rpd3Δ/rpd3Δ strains for their effects on spontaneous mitotic recombination. By assaying a variety of intra- and inter-genic recombination events distributed over three chromosomes, we found that in the majority of cases spontaneous mitotic recombination was reduced in diploid rpd3Δ/rpd3Δ cells (relative to a RPD3/RPD3 control). Finally, although 90% of mitotic recombinant events are initiated in the G₁ phase of the growth cycle (i.e., before DNA synthesis) we show that RPD3 is not regulated in a cell-cycle-dependent manner. These data suggest that mitotic recombination, in addition to gene expression, is affected by changes in chromatin architecture mediated by RPD3. Received: 17 July / 30 November 1998     

Synergism between yeast nucleotide and base excision repair pathways in the protection against DNA methylation damage

The treatment of cells with simple DNA methylating agents such as methyl methanesulfonate (MMS) results in genotoxic lesions, including 3-methyladenine which blocks DNA replication. All the organisms studied to date contain an alkylation-specific base excision repair pathway. In the yeast Saccharomyces cerevisiae, the base excision repair pathway is initiated by a Mag1 3-methyladenine DNA glycosylase that removes the damaged base, followed by the Apn1 apurinic/apyrimidinic endonuclease which cleaves the DNA strand at the abasic site for subsequent repair and synthesis. Several nucleotide excision repair pathway mutants display only slightly increased sensitivity to killing by MMS, indicating that nucleotide excision repair per se does not play a major role in the repair of DNA methylation damage. However, mag1 and apn1 mutants that are also defective in nucleotide excision repair are extremely sensitive to MMS-induced killing and the effects are synergistic. These observations suggest that nucleotide excision repair and alkylation-specific base excision repair provide alternative pathways for the repair of DNA methylation damage. In addition to their role in nucleotide excision repair, Rad1 and Rad10 form a complex that is involved in recombination repair. It was found that the apn1 rad1 and apn1 rad10 double mutants have a growth defect and are significantly more sensitive to MMS killing than apn1 rad2 and apn1 rad4 double mutants in a gradient plate assay. Furthermore, the apn1 rad1 double mutant increased both the spontaneous and MMS-induced mutation frequency. Thus, the recombination repair defects of rad1 and rad10 may confer an additional synergistic effect when combined with the apn1 mutation. Received: 8 September 1997 / 13 November 1997     

Spontaneous mitotic recombination inSchizosaccharomyces pomhe

Summary The UV sensitive mutantrad2-44 ofSchizosaccharomyces pomhe increases mitotic gene conversion and crossover rates about 10-fold but has little or no effect on meiotic recombination. As inrad2⁺, recombination events on different chromosomes are coincidental inrad2-44, indicating that mitotic recombination takes place in a subpopulation of competent cells. However, the coefficient of coincidence is smaller in the mutant, whereas recombination rates among the competent cells are the same as inrad2⁺. This suggests thatrad2-44 increases mitotic recombination by enhancing the fraction of competent cells. The rate limiting factor in spontaneous mitotic recombination inS. pomhe appears to be the size of the subpopulation of recombinationally competent cells.     

Evidence that the single CDC8 gene which encodes thymidylate kinase is involved in DNA replication,recombination and mutation in yeast

Summary The conditional cdc8 mutant is known to be defective, under restrictive conditions, in the elongation of DNA during synthesis. In yeast the CDC8 gene encodes thymidylate kinase. We show here that UV-induced gene conversion and gene mutation events require the participation of this CDC8 gene. Thus, the same thymidylate kinase is incolved both in DNA replication and in UV-induced gene conversion and gene mutation in yeast.     

DNA repair and recombination functions in <Emphasis Type="Italic">Arabidopsis</Emphasis> telomere maintenance

In this review, we discuss recent advances in the knowledge of plant telomere maintenance, focusing on the model plant Arabidopsis thaliana and, in particular, on the roles of proteins involved in DNA repair and recombination. The question of the interrelationships between DNA repair and recombination pathways and proteins with telomere function and maintenance is of increasing interest and has been the subject of a number of recent reviews (Cech 2004, d’Adda di Fagagna et al. 2004, Hande 2004, Harrington 2004, Maser & DePinho 2004). Understanding of telomere biology, DNA repair and recombination in plants has rapidly progressed over the last decade, substantially due to genetic approaches in Arabidopsis, and we feel that this is an appropriate time to review current knowledge in this field. A number of recent reviews have dealt more generally with the subject of plant telomere structure and evolution (Riha et al. 2001, McKnight et al. 2002, Riha & Shippen 2003b, McKnight & Shippen 2004, Fajkus et al. 2005) and we thus focus specifically on plant telomere biology in the context of DNA repair and recombination in Arabidopsis.     

Exposure of cdc8 homozygous diploids of Saccharomyces cerevisiae to nonpermissive temperature leads to an increase in mitotic conversion frequencies

Summary In a diploid strain homozygous for the cdc8-1 mutation, a block in DNA synthesis caused by restrictive temperature resulted in a significant increase in the frequency of intragenic recombination at the HOM2 locus. Under restrictive conditions, incorporation of radioactivity into DNA was reduced to 2% of the control and alkaline sucrose gradient centrifugation revealed that only short DNA fragments were synthesized. There was no considerable fragmentation of template DNA during incubation of cdc8-1 strains under restrictive conditions.     

Mitotic recombination in stable and unstable chromosome III disomics

Summary Approximately 2% of the haploid breakdown sectors of heterozygous chromosome III disomics of Aspergillus nidulans are the result of recombination between the homologous chromosomes. The exchanges are concentrated between the two mutations spanning the centromere. Comparisons are made between disomics hemizygous for the sod ^III A1 mutation (Upshall et al. 1979) which are stable when grown at 37 °C, and disomics carrying the wild type allele of the sod ^IIIA1 locus, which are unstable under all conditions. It is shown that neither temperature nor the sod ^IIIA1 mutation affect the frequency or pattern of recombination between the homologues.     

Nonrandomly-associated forward mutation and mitotic recombination yield yeast diploids homozygous for recessive mutations

We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L--aminoadipate as a nitrogen source (-Aa⁺) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. -Aa^- diploid cells yield -Aa⁺ mutants at a rate of 7.8±3.6×10^-9. As in haploid strains, approximately 97% (30/31) of -Aa⁺ mutants are spontaneous lys2-x recessive mutations. -Aa⁺ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.2±0.4×10^-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.3±0.6×10^-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene conversion of LYS2 to lys2-x; 85% of lys2-x/lys2-x diploids appear to have arisen by mitotic recombination in the CENII-LYS2 interval. lys2-1/lys2-1 mitotic segregants of a control LYS2/lys2-1 diploid consist similarly of 18% of lys2-1/lys2-1 diploids that appear to have arisen by gene conversion of LYS2 to lys2-1 and 82% of lys2-1/lys2-1 diploids that appear to have arisen by mitotic recombination in the CENII-LYS2 interval. The methods described can be used to simultaneously monitor the effects of yeast gene mutations and carcinogens on the principal parameters affecting the genomic stability of diploid mitotic cells: mutation, gene conversion, intergenic recombination, and chromosomal loss or rearrangement.The research of the authors was supported by the Director, Office of Energy Research, Biological Research Division of the U.S. Department of Energy under Contract No. DE-ACO3-76SF00098, grants to M. S. E. and C. V. B. from the National Institutes of Health and the National Aeronautics and Space Administration, and a National Science Foundation postdoctoral research fellowship award to R. M. R.     

The Escherichia coli recA gene increases UV-induced mitotic gene conversion in Saccharomyces cerevisiae

The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceeding mitotic conversion events in yeast.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Genetic and molecular analysis of REC114, an early meiotic recombination gene in yeast

Four new meiotic recombination genes were previously isolated by selecting for mutations that rescue the meiotic lethality of rad52 spo13 strains. One of these genes, REC114, is described here, and the data confirm that REC114 is a meiosis-specific recombination gene with no detectable function in mitosis. REC114 is located on chromosome XIII approximately 4,9 cM from CIN4. The nucleotide sequence reveals an open reading frame of 1262 bp, consensus intron splice sites close to the 3 end, and indicates that the second exon codes for only seven amino acids. In the promoter region, a URS1 consensus sequence (TGGGCGGCTA), identical to the URS1 found in the promoter of SPO16, is present 93 bp upstream of the translation start site. Northern-blot hybridization demonstrates that REC114 is transcribed only during meiosis and that it is not expressed in the absence of the IME1 gene product, even when IME2 is constitutively expressed.     

Cloning of photoreactivation repair gene and excision repair gene of the yeast Saccharomyces cerevisiae

Summary The photoreactivation repair gene (PHR1) of the yeast Saccharomyces cerevisiae was cloned in a hybrid plasmid (pJDB207), which is able to replicate as a multicopy episome in S. cerevisiae and Escherichia coli cells. The size of the DNA fragment found to have the photoreactivation activity was 3.0 kb, determined by recloning of the isolated fragment. In wild type cells transformed by the plasmid containing the PHR1 gene, the number of DNA photolyase molecules was 15 times greater than in wild type cells with pJDB207 only. Using the same receptor strain the excision repair gen RAD1 was also isolated. The size of the insert of the DNA which complements excision repair deficiency in recipient yeast cells was 5.7 kb. The recipient cells after transformation with the plasmid containing RAD1 showed the same UV-sensitivty as wild type cells with pJDB207 only.Abbreviation UV Ultra-violet light of 254 run wavelength     

The mcm2-1 mutation of yeast causes DNA damage with a RAD9 requirement for repair

The minichromosome maintenance mutation, mcm2-1, has been found to synthesize damaged DNA at 35°C. Growth at this temperature rendered the mutant strain more sensitive to killing by ultraviolet irradiation. DNA damage could also be detected by pulsed-field gel electrophoresis, where a higher fraction of the DNA loaded was retained in the inserts at the wells. During the exponential phase of growth at this temperature about 50% of the cells had large buds, with the nucleus at or near the neck of the bud in most cases. The incorporation of the rad9 deletion in the mcm2-1-carrying strain caused a reduction in the percentage of large-budded cells and a moderate loss of cell viability. The results are consistent with mcm2-1 causing DNA damage leading to the arrest of cells in the S/G₂ phase of the cell cycle, which was partially dependent on the RAD9 gene product.     

DNA damage repair response in mesenchymal stromal cells: From cellular senescence and aging to apoptosis and differentiation ability

Mesenchymal stromal cells (MSCs) are heterogeneous and contain several populations, including stem cells. MSCs' secretome has the ability to induce proliferation, differentiation, chemo-attraction, anti-apoptosis, and immunomodulation activities in stem cells. Moreover, these cells recognize tissue damage caused by drugs, radiation (e.g., Ultraviolet, infra-red) and oxidative stress, and respond in two ways: either MSCs differentiate into particular cell lineages to preserve tissue homeostasis, or they release a regenerative secretome to activate tissue repairing mechanisms. The maintenance of MSCs in quiescence can increase the incidence and accumulation of various forms of genomic modifications, particularly upon environmental insults. Thus, dysregulated DNA repair pathways can predispose MSCs to senescence or apoptosis, reducing their stemness and self-renewal properties. For instance, DNA damage can impair telomere replication, activating DNA damage checkpoints to maintain MSC function. In this review, we aim to summarize the role of DNA damage and associated repair responses in MSC senescence, differentiation and programmed cell death.     

Unequal sister-strand recombination within yeast ribosomal DNA does not require the RAD 52 gene product

Summary We have found that the RAD52 gene product, which is required for gene conversion and recombination in the yeast Saccharomyces cerevisiae, is not required for unequal mitotic sister-strand recombination.     

Construction of a marker gene cassette which is repeatedly usable for gene disruption in yeast

A disruption cassette has been constructed containing the LEU2 gene flanked by directly repeated sitespecific recombination sites of the yeast plasmid, pSB3, which resembles the 2 m DNA of Saccharomyces cerevisiae. A disruption constructed by inserting this DNA fragment acquires a Leu⁺ phenotype, which can be easily removed by expressing the FLP-PSB3 gene encoding the site-specific recombinase of pSB3. A test was made using a Schizosaccharomyces pombe host. The ura4 ⁺ gene of S. pombe was replaced with the ura4::LEU2 gene constructed by inserting the disruption cassette into the ura4 ⁺ gene. Then, the FLP-pSB3 gene driven by the nmt1 ⁺ promoter was introduced into this disruptant. Upon de-repression of the nmt1 promoter by removing thiamine from the medium, the rate of appearance of Leu^- was increased. As expected the ura4 ⁺ locus underwent a structural change. Thus, the FLP-pSB3 protein and its target site can function adequately in S. pombe.