Abstract
In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.
Full Text
The Full Text of this article is available as a PDF (239.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adzuma K., Ogawa T., Ogawa H. Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2735–2744. doi: 10.1128/mcb.4.12.2735. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ajima Jun, Umezu Keiko, Maki Hisaji. Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy, interchromosomal rearrangement, and the simultaneous incidence of both events during mitotic growth. Mutat Res. 2002 Jul 25;504(1-2):157–172. doi: 10.1016/s0027-5107(02)00089-1. [DOI] [PubMed] [Google Scholar]
- Alani E., Cao L., Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987 Aug;116(4):541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Alani E., Subbiah S., Kleckner N. The yeast RAD50 gene encodes a predicted 153-kD protein containing a purine nucleotide-binding domain and two large heptad-repeat regions. Genetics. 1989 May;122(1):47–57. doi: 10.1093/genetics/122.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aström S. U., Okamura S. M., Rine J. Yeast cell-type regulation of DNA repair. Nature. 1999 Jan 28;397(6717):310–310. doi: 10.1038/16833. [DOI] [PubMed] [Google Scholar]
- Bai Y., Symington L. S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 1996 Aug 15;10(16):2025–2037. doi: 10.1101/gad.10.16.2025. [DOI] [PubMed] [Google Scholar]
- Bennett C. B., Lewis L. K., Karthikeyan G., Lobachev K. S., Jin Y. H., Sterling J. F., Snipe J. R., Resnick M. A. Genes required for ionizing radiation resistance in yeast. Nat Genet. 2001 Dec;29(4):426–434. doi: 10.1038/ng778. [DOI] [PubMed] [Google Scholar]
- Boulton S. J., Jackson S. P. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 1998 Mar 16;17(6):1819–1828. doi: 10.1093/emboj/17.6.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bressan D. A., Baxter B. K., Petrini J. H. The Mre11-Rad50-Xrs2 protein complex facilitates homologous recombination-based double-strand break repair in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Nov;19(11):7681–7687. doi: 10.1128/mcb.19.11.7681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bressan D. A., Olivares H. A., Nelms B. E., Petrini J. H. Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11. Genetics. 1998 Oct;150(2):591–600. doi: 10.1093/genetics/150.2.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen L., Trujillo K., Ramos W., Sung P., Tomkinson A. E. Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol Cell. 2001 Nov;8(5):1105–1115. doi: 10.1016/s1097-2765(01)00388-4. [DOI] [PubMed] [Google Scholar]
- Cox M. M., Goodman M. F., Kreuzer K. N., Sherratt D. J., Sandler S. J., Marians K. J. The importance of repairing stalled replication forks. Nature. 2000 Mar 2;404(6773):37–41. doi: 10.1038/35003501. [DOI] [PubMed] [Google Scholar]
- De Antoni A., Gallwitz D. A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae. Gene. 2000 Apr 4;246(1-2):179–185. doi: 10.1016/s0378-1119(00)00083-4. [DOI] [PubMed] [Google Scholar]
- Galgoczy D. J., Toczyski D. P. Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast. Mol Cell Biol. 2001 Mar;21(5):1710–1718. doi: 10.1128/MCB.21.5.1710-1718.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grenon M., Gilbert C., Lowndes N. F. Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat Cell Biol. 2001 Sep;3(9):844–847. doi: 10.1038/ncb0901-844. [DOI] [PubMed] [Google Scholar]
- Heude M., Fabre F. a/alpha-control of DNA repair in the yeast Saccharomyces cerevisiae: genetic and physiological aspects. Genetics. 1993 Mar;133(3):489–498. doi: 10.1093/genetics/133.3.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hiraoka M., Watanabe K., Umezu K., Maki H. Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells. Genetics. 2000 Dec;156(4):1531–1548. doi: 10.1093/genetics/156.4.1531. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hopfner K. P., Karcher A., Craig L., Woo T. T., Carney J. P., Tainer J. A. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell. 2001 May 18;105(4):473–485. doi: 10.1016/s0092-8674(01)00335-x. [DOI] [PubMed] [Google Scholar]
- Ivanov E. L., Korolev V. G., Fabre F. XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. Genetics. 1992 Nov;132(3):651–664. doi: 10.1093/genetics/132.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ivanov E. L., Sugawara N., White C. I., Fabre F., Haber J. E. Mutations in XRS2 and RAD50 delay but do not prevent mating-type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1994 May;14(5):3414–3425. doi: 10.1128/mcb.14.5.3414. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kadyk L. C., Hartwell L. H. Replication-dependent sister chromatid recombination in rad1 mutants of Saccharomyces cerevisiae. Genetics. 1993 Mar;133(3):469–487. doi: 10.1093/genetics/133.3.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kadyk L. C., Hartwell L. H. Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. Genetics. 1992 Oct;132(2):387–402. doi: 10.1093/genetics/132.2.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kagawa W., Kurumizaka H., Ikawa S., Yokoyama S., Shibata T. Homologous pairing promoted by the human Rad52 protein. J Biol Chem. 2001 Jul 13;276(37):35201–35208. doi: 10.1074/jbc.M104938200. [DOI] [PubMed] [Google Scholar]
- Klein H. L. Genetic control of intrachromosomal recombination. Bioessays. 1995 Feb;17(2):147–159. doi: 10.1002/bies.950170210. [DOI] [PubMed] [Google Scholar]
- Klein H. L. RDH54, a RAD54 homologue in Saccharomyces cerevisiae, is required for mitotic diploid-specific recombination and repair and for meiosis. Genetics. 1997 Dec;147(4):1533–1543. doi: 10.1093/genetics/147.4.1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee S. E., Moore J. K., Holmes A., Umezu K., Kolodner R. D., Haber J. E. Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell. 1998 Aug 7;94(3):399–409. doi: 10.1016/s0092-8674(00)81482-8. [DOI] [PubMed] [Google Scholar]
- Liefshitz B., Steinlauf R., Friedl A., Eckardt-Schupp F., Kupiec M. Genetic interactions between mutants of the 'error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis. Mutat Res. 1998 Mar;407(2):135–145. doi: 10.1016/s0921-8777(97)00070-0. [DOI] [PubMed] [Google Scholar]
- Malone R. E., Ward T., Lin S., Waring J. The RAD50 gene, a member of the double strand break repair epistasis group, is not required for spontaneous mitotic recombination in yeast. Curr Genet. 1990 Aug;18(2):111–116. doi: 10.1007/BF00312598. [DOI] [PubMed] [Google Scholar]
- Morrison D. P., Hastings P. J. Characterization of the mutator mutation mut5-1. Mol Gen Genet. 1979 Aug;175(1):57–65. doi: 10.1007/BF00267856. [DOI] [PubMed] [Google Scholar]
- Mortimer R. K., Contopoulou R., Schild D. Mitotic chromosome loss in a radiation-sensitive strain of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5778–5782. doi: 10.1073/pnas.78.9.5778. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ogawa T., Shinohara A., Nabetani A., Ikeya T., Yu X., Egelman E. H., Ogawa H. RecA-like recombination proteins in eukaryotes: functions and structures of RAD51 genes. Cold Spring Harb Symp Quant Biol. 1993;58:567–576. doi: 10.1101/sqb.1993.058.01.063. [DOI] [PubMed] [Google Scholar]
- Paulovich A. G., Armour C. D., Hartwell L. H. The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage. Genetics. 1998 Sep;150(1):75–93. doi: 10.1093/genetics/150.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pâques F., Haber J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999 Jun;63(2):349–404. doi: 10.1128/mmbr.63.2.349-404.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roche H., Gietz R. D., Kunz B. A. Specificities of the Saccharomyces cerevisiae rad6, rad18, and rad52 mutators exhibit different degrees of dependence on the REV3 gene product, a putative nonessential DNA polymerase. Genetics. 1995 Jun;140(2):443–456. doi: 10.1093/genetics/140.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shibata T., Nishinaka T., Mikawa T., Aihara H., Kurumizaka H., Yokoyama S., Ito Y. Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: a possible advantage of DNA over RNA as genomic material. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8425–8432. doi: 10.1073/pnas.111005198. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shinohara A., Ogawa H., Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–470. doi: 10.1016/0092-8674(92)90447-k. [DOI] [PubMed] [Google Scholar]
- Signon L., Malkova A., Naylor M. L., Klein H., Haber J. E. Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break. Mol Cell Biol. 2001 Mar;21(6):2048–2056. doi: 10.1128/MCB.21.6.2048-2056.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sung P., Trujillo K. M., Van Komen S. Recombination factors of Saccharomyces cerevisiae. Mutat Res. 2000 Jun 30;451(1-2):257–275. doi: 10.1016/s0027-5107(00)00054-3. [DOI] [PubMed] [Google Scholar]
- Tsubouchi H., Ogawa H. A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. Mol Cell Biol. 1998 Jan;18(1):260–268. doi: 10.1128/mcb.18.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Umezu Keiko, Hiraoka Mina, Mori Masaaki, Maki Hisaji. Structural analysis of aberrant chromosomes that occur spontaneously in diploid Saccharomyces cerevisiae: retrotransposon Ty1 plays a crucial role in chromosomal rearrangements. Genetics. 2002 Jan;160(1):97–110. doi: 10.1093/genetics/160.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Usui T., Ogawa H., Petrini J. H. A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol Cell. 2001 Jun;7(6):1255–1266. doi: 10.1016/s1097-2765(01)00270-2. [DOI] [PubMed] [Google Scholar]
- Watanabe Keiichi, Morishita Jun, Umezu Keiko, Shirahige Katsuhiko, Maki Hisaji. Involvement of RAD9-dependent damage checkpoint control in arrest of cell cycle, induction of cell death, and chromosome instability caused by defects in origin recognition complex in Saccharomyces cerevisiae. Eukaryot Cell. 2002 Apr;1(2):200–212. doi: 10.1128/EC.1.2.200-212.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Jager M., van Noort J., van Gent D. C., Dekker C., Kanaar R., Wyman C. Human Rad50/Mre11 is a flexible complex that can tether DNA ends. Mol Cell. 2001 Nov;8(5):1129–1135. doi: 10.1016/s1097-2765(01)00381-1. [DOI] [PubMed] [Google Scholar]