Abstract
We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB) -induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence.
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- Aguilera A., Klein H. L. Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics. 1988 Aug;119(4):779–790. doi: 10.1093/genetics/119.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aguilera A., Klein H. L. Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products. Genetics. 1989 Dec;123(4):683–694. doi: 10.1093/genetics/123.4.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ahn B. Y., Livingston D. M. Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system. Mol Cell Biol. 1986 Nov;6(11):3685–3693. doi: 10.1128/mcb.6.11.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Albertini A. M., Hofer M., Calos M. P., Miller J. H. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. doi: 10.1016/0092-8674(82)90148-9. [DOI] [PubMed] [Google Scholar]
- Bailly V., Sommers C. H., Sung P., Prakash L., Prakash S. Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8273–8277. doi: 10.1073/pnas.89.17.8273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baltimore D. Gene conversion: some implications for immunoglobulin genes. Cell. 1981 Jun;24(3):592–594. doi: 10.1016/0092-8674(81)90082-9. [DOI] [PubMed] [Google Scholar]
- Bardwell L., Cooper A. J., Friedberg E. C. Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro. Mol Cell Biol. 1992 Jul;12(7):3041–3049. doi: 10.1128/mcb.12.7.3041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baur M., Potrykus I., Paszkowski J. Intermolecular homologous recombination in plants. Mol Cell Biol. 1990 Feb;10(2):492–500. doi: 10.1128/mcb.10.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Belmaaza A., Chartrand P. One-sided invasion events in homologous recombination at double-strand breaks. Mutat Res. 1994 May;314(3):199–208. doi: 10.1016/0921-8777(94)90065-5. [DOI] [PubMed] [Google Scholar]
- Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
- Bollag R. J., Waldman A. S., Liskay R. M. Homologous recombination in mammalian cells. Annu Rev Genet. 1989;23:199–225. doi: 10.1146/annurev.ge.23.120189.001215. [DOI] [PubMed] [Google Scholar]
- Calderón I. L., Cerdá-Olmedo E. Induction by N-methyl-N'-nitro-N-nitrosoguanidine of nuclear and cytoplasmic mutations in Saccharomyces cerevisiae. Mutat Res. 1983 Mar;108(1-3):133–146. doi: 10.1016/0027-5107(83)90115-x. [DOI] [PubMed] [Google Scholar]
- Chan C. S., Botstein D. Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast. Genetics. 1993 Nov;135(3):677–691. doi: 10.1093/genetics/135.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dornfeld K. J., Livingston D. M. Plasmid recombination in a rad52 mutant of Saccharomyces cerevisiae. Genetics. 1992 Jun;131(2):261–276. doi: 10.1093/genetics/131.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
- Fishman-Lobell J., Haber J. E. Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science. 1992 Oct 16;258(5081):480–484. doi: 10.1126/science.1411547. [DOI] [PubMed] [Google Scholar]
- Fishman-Lobell J., Rudin N., Haber J. E. Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol Cell Biol. 1992 Mar;12(3):1292–1303. doi: 10.1128/mcb.12.3.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Friedberg E. C. Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae. Microbiol Rev. 1988 Mar;52(1):70–102. doi: 10.1128/mr.52.1.70-102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fukuchi K., Martin G. M., Monnat R. J., Jr Mutator phenotype of Werner syndrome is characterized by extensive deletions. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5893–5897. doi: 10.1073/pnas.86.15.5893. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gordenin D. A., Malkova A. L., Peterzen A., Kulikov V. N., Pavlov Y. I., Perkins E., Resnick M. A. Transposon Tn5 excision in yeast: influence of DNA polymerases alpha, delta, and epsilon and repair genes. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3785–3789. doi: 10.1073/pnas.89.9.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henderson S. T., Petes T. D. Instability of simple sequence DNA in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Jun;12(6):2749–2757. doi: 10.1128/mcb.12.6.2749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
- Jackson J. A., Fink G. R. Gene conversion between duplicated genetic elements in yeast. Nature. 1981 Jul 23;292(5821):306–311. doi: 10.1038/292306a0. [DOI] [PubMed] [Google Scholar]
- Jinks-Robertson S., Michelitch M., Ramcharan S. Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Jul;13(7):3937–3950. doi: 10.1128/mcb.13.7.3937. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaytor M. D., Livingston D. M. Saccharomyces cerevisiae RAD52 alleles temperature-sensitive for the repair of DNA double-strand breaks. Genetics. 1994 Aug;137(4):933–944. doi: 10.1093/genetics/137.4.933. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klein H. L. Different types of recombination events are controlled by the RAD1 and RAD52 genes of Saccharomyces cerevisiae. Genetics. 1988 Oct;120(2):367–377. doi: 10.1093/genetics/120.2.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klein H. L. Lack of association between intrachromosomal gene conversion and reciprocal exchange. 1984 Aug 30-Sep 5Nature. 310(5980):748–753. doi: 10.1038/310748a0. [DOI] [PubMed] [Google Scholar]
- Klein H. L., Petes T. D. Intrachromosomal gene conversion in yeast. Nature. 1981 Jan 15;289(5794):144–148. doi: 10.1038/289144a0. [DOI] [PubMed] [Google Scholar]
- Larionov V., Kouprina N., Eldarov M., Perkins E., Porter G., Resnick M. A. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks. Yeast. 1994 Jan;10(1):93–104. doi: 10.1002/yea.320100109. [DOI] [PubMed] [Google Scholar]
- Liskay R. M., Stachelek J. L. Evidence for intrachromosomal gene conversion in cultured mouse cells. Cell. 1983 Nov;35(1):157–165. doi: 10.1016/0092-8674(83)90218-0. [DOI] [PubMed] [Google Scholar]
- Lovett S. T., Drapkin P. T., Sutera V. A., Jr, Gluckman-Peskind T. J. A sister-strand exchange mechanism for recA-independent deletion of repeated DNA sequences in Escherichia coli. Genetics. 1993 Nov;135(3):631–642. doi: 10.1093/genetics/135.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maloney D. H., Fogel S. Gene conversion, unequal crossing-over and mispairing at a non-tandem duplication during meiosis of Saccharomyces cerevisiae. Curr Genet. 1987;12(1):1–7. doi: 10.1007/BF00420720. [DOI] [PubMed] [Google Scholar]
- Maryon E., Carroll D. Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination. Mol Cell Biol. 1991 Jun;11(6):3278–3287. doi: 10.1128/mcb.11.6.3278. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mazin A. V., Kuzminov A. V., Dianov G. L., Salganik R. I. Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats. Mol Gen Genet. 1991 Aug;228(1-2):209–214. doi: 10.1007/BF00282467. [DOI] [PubMed] [Google Scholar]
- Meyn M. S. High spontaneous intrachromosomal recombination rates in ataxia-telangiectasia. Science. 1993 May 28;260(5112):1327–1330. doi: 10.1126/science.8493577. [DOI] [PubMed] [Google Scholar]
- Murray A. W., Szostak J. W. Pedigree analysis of plasmid segregation in yeast. Cell. 1983 Oct;34(3):961–970. doi: 10.1016/0092-8674(83)90553-6. [DOI] [PubMed] [Google Scholar]
- Nickoloff J. A., Singer J. D., Hoekstra M. F., Heffron F. Double-strand breaks stimulate alternative mechanisms of recombination repair. J Mol Biol. 1989 Jun 5;207(3):527–541. doi: 10.1016/0022-2836(89)90462-2. [DOI] [PubMed] [Google Scholar]
- Ozenberger B. A., Roeder G. S. A unique pathway of double-strand break repair operates in tandemly repeated genes. Mol Cell Biol. 1991 Mar;11(3):1222–1231. doi: 10.1128/mcb.11.3.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petes T. D., Hill C. W. Recombination between repeated genes in microorganisms. Annu Rev Genet. 1988;22:147–168. doi: 10.1146/annurev.ge.22.120188.001051. [DOI] [PubMed] [Google Scholar]
- Prado F., Aguilera A. New in-vivo cloning methods by homologous recombination in yeast. Curr Genet. 1994 Feb;25(2):180–183. doi: 10.1007/BF00309546. [DOI] [PubMed] [Google Scholar]
- Ronne H., Rothstein R. Mitotic sectored colonies: evidence of heteroduplex DNA formation during direct repeat recombination. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2696–2700. doi: 10.1073/pnas.85.8.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rudin N., Sugarman E., Haber J. E. Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics. 1989 Jul;122(3):519–534. doi: 10.1093/genetics/122.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruskin B., Fink G. R. Mutations in POL1 increase the mitotic instability of tandem inverted repeats in Saccharomyces cerevisiae. Genetics. 1993 May;134(1):43–56. doi: 10.1093/genetics/134.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
- Schiestl R. H., Prakash S. RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination. Mol Cell Biol. 1988 Sep;8(9):3619–3626. doi: 10.1128/mcb.8.9.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schiestl R. H., Prakash S. RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination. Mol Cell Biol. 1990 Jun;10(6):2485–2491. doi: 10.1128/mcb.10.6.2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Strand M., Prolla T. A., Liskay R. M., Petes T. D. Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature. 1993 Sep 16;365(6443):274–276. doi: 10.1038/365274a0. [DOI] [PubMed] [Google Scholar]
- Sugawara N., Haber J. E. Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation. Mol Cell Biol. 1992 Feb;12(2):563–575. doi: 10.1128/mcb.12.2.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sung P., Prakash L., Prakash S. Renaturation of DNA catalysed by yeast DNA repair and recombination protein RAD10. Nature. 1992 Feb 20;355(6362):743–745. doi: 10.1038/355743a0. [DOI] [PubMed] [Google Scholar]
- Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
- Takahashi N. K., Yamamoto K., Kitamura Y., Luo S. Q., Yoshikura H., Kobayashi I. Nonconservative recombination in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5912–5916. doi: 10.1073/pnas.89.13.5912. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thibodeau S. N., Bren G., Schaid D. Microsatellite instability in cancer of the proximal colon. Science. 1993 May 7;260(5109):816–819. doi: 10.1126/science.8484122. [DOI] [PubMed] [Google Scholar]
- Thomas B. J., Rothstein R. The genetic control of direct-repeat recombination in Saccharomyces: the effect of rad52 and rad1 on mitotic recombination at GAL10, a transcriptionally regulated gene. Genetics. 1989 Dec;123(4):725–738. doi: 10.1093/genetics/123.4.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tomkinson A. E., Bardwell A. J., Bardwell L., Tappe N. J., Friedberg E. C. Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease. Nature. 1993 Apr 29;362(6423):860–862. doi: 10.1038/362860a0. [DOI] [PubMed] [Google Scholar]
- Willis K. K., Klein H. L. Intrachromosomal recombination in Saccharomyces cerevisiae: reciprocal exchange in an inverted repeat and associated gene conversion. Genetics. 1987 Dec;117(4):633–643. doi: 10.1093/genetics/117.4.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yuan L. W., Keil R. L. Distance-independence of mitotic intrachromosomal recombination in Saccharomyces cerevisiae. Genetics. 1990 Feb;124(2):263–273. doi: 10.1093/genetics/124.2.263. [DOI] [PMC free article] [PubMed] [Google Scholar]