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. 1988 Sep;8(9):3918–3928. doi: 10.1128/mcb.8.9.3918

Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences.

N Rudin 1, J E Haber 1
PMCID: PMC365451  PMID: 3065627

Abstract

Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated. When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats. This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB. Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time. Repair is dependent on the RAD52 gene. The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage. By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction. Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  2. Budd M., Mortimer R. K. The effect of cycloheximide on repair in a temperature conditional radiation-sensitive mutant of Saccharomyces cerevisiae. Radiat Res. 1984 Sep;99(3):582–590. [PubMed] [Google Scholar]
  3. Cole G. M., Schild D., Lovett S. T., Mortimer R. K. Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage. Mol Cell Biol. 1987 Mar;7(3):1078–1084. doi: 10.1128/mcb.7.3.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Connolly B., White C. I., Haber J. E. Physical monitoring of mating type switching in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jun;8(6):2342–2349. doi: 10.1128/mcb.8.6.2342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dunn B., Szauter P., Pardue M. L., Szostak J. W. Transfer of yeast telomeres to linear plasmids by recombination. Cell. 1984 Nov;39(1):191–201. doi: 10.1016/0092-8674(84)90205-8. [DOI] [PubMed] [Google Scholar]
  6. Game J. C., Zamb T. J., Braun R. J., Resnick M., Roth R. M. The Role of Radiation (rad) Genes in Meiotic Recombination in Yeast. Genetics. 1980 Jan;94(1):51–68. doi: 10.1093/genetics/94.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Greider C. W., Blackburn E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405–413. doi: 10.1016/0092-8674(85)90170-9. [DOI] [PubMed] [Google Scholar]
  8. Guarente L., Yocum R. R., Gifford P. A GAL10-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7410–7414. doi: 10.1073/pnas.79.23.7410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haber J. E., Hearn M. Rad52-independent mitotic gene conversion in Saccharomyces cerevisiae frequently results in chromosomal loss. Genetics. 1985 Sep;111(1):7–22. doi: 10.1093/genetics/111.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haber J. E., Thorburn P. C. Healing of broken linear dicentric chromosomes in yeast. Genetics. 1984 Feb;106(2):207–226. doi: 10.1093/genetics/106.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hereford L. M., Hartwell L. H. Role of protein synthesis in the replication of yeast DNA. Nat New Biol. 1973 Aug 1;244(135):129–131. doi: 10.1038/newbio244129a0. [DOI] [PubMed] [Google Scholar]
  12. Hicks J. B., Herskowitz I. Interconversion of Yeast Mating Types I. Direct Observations of the Action of the Homothallism (HO) Gene. Genetics. 1976 Jun;83(2):245–258. doi: 10.1093/genetics/83.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Klar A. J., Hicks J. B., Strathern J. N. Directionality of yeast mating-type interconversion. Cell. 1982 Mar;28(3):551–561. doi: 10.1016/0092-8674(82)90210-0. [DOI] [PubMed] [Google Scholar]
  16. Klar A. J., Strathern J. N., Abraham J. A. Involvement of double-strand chromosomal breaks for mating-type switching in Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 1984;49:77–88. doi: 10.1101/sqb.1984.049.01.011. [DOI] [PubMed] [Google Scholar]
  17. Klar A. J., Strathern J. N. Resolution of recombination intermediates generated during yeast mating type switching. 1984 Aug 30-Sep 5Nature. 310(5980):744–748. doi: 10.1038/310744a0. [DOI] [PubMed] [Google Scholar]
  18. Kolodkin A. L., Klar A. J., Stahl F. W. Double-strand breaks can initiate meiotic recombination in S. cerevisiae. Cell. 1986 Aug 29;46(5):733–740. doi: 10.1016/0092-8674(86)90349-1. [DOI] [PubMed] [Google Scholar]
  19. Kostriken R., Heffron F. The product of the HO gene is a nuclease: purification and characterization of the enzyme. Cold Spring Harb Symp Quant Biol. 1984;49:89–96. doi: 10.1101/sqb.1984.049.01.012. [DOI] [PubMed] [Google Scholar]
  20. Kostriken R., Strathern J. N., Klar A. J., Hicks J. B., Heffron F. A site-specific endonuclease essential for mating-type switching in Saccharomyces cerevisiae. Cell. 1983 Nov;35(1):167–174. doi: 10.1016/0092-8674(83)90219-2. [DOI] [PubMed] [Google Scholar]
  21. Kunes S., Botstein D., Fox M. S. Transformation of yeast with linearized plasmid DNA. Formation of inverted dimers and recombinant plasmid products. J Mol Biol. 1985 Aug 5;184(3):375–387. doi: 10.1016/0022-2836(85)90288-8. [DOI] [PubMed] [Google Scholar]
  22. Lichten M., Borts R. H., Haber J. E. Meiotic gene conversion and crossing over between dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae. Genetics. 1987 Feb;115(2):233–246. doi: 10.1093/genetics/115.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Malone R. E., Esposito R. E. The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast. Proc Natl Acad Sci U S A. 1980 Jan;77(1):503–507. doi: 10.1073/pnas.77.1.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCusker J. H., Haber J. E. Evidence of Chromosomal Breaks near the Mating-Type Locus of SACCHAROMYCES CEREVISIAE That Accompany MATalpha xMATalpha Matings. Genetics. 1981 Nov;99(3-4):383–403. doi: 10.1093/genetics/99.3-4.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Nasmyth K. A. Molecular genetics of yeast mating type. Annu Rev Genet. 1982;16:439–500. doi: 10.1146/annurev.ge.16.120182.002255. [DOI] [PubMed] [Google Scholar]
  27. Nasmyth K. Molecular analysis of a cell lineage. Nature. 1983 Apr 21;302(5910):670–676. doi: 10.1038/302670a0. [DOI] [PubMed] [Google Scholar]
  28. Nickoloff J. A., Chen E. Y., Heffron F. A 24-base-pair DNA sequence from the MAT locus stimulates intergenic recombination in yeast. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7831–7835. doi: 10.1073/pnas.83.20.7831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Orr-Weaver T. L., Szostak J. W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417–4421. doi: 10.1073/pnas.80.14.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oshima T., Takano I. Mutants Showing Heterothallism from a Homothallic Strain of SACCHAROMYCES CEREVISIAE. Genetics. 1980 Apr;94(4):841–857. doi: 10.1093/genetics/94.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Prakash S., Prakash L., Burke W., Montelone B. A. Effects of the RAD52 Gene on Recombination in SACCHAROMYCES CEREVISIAE. Genetics. 1980 Jan;94(1):31–50. doi: 10.1093/genetics/94.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Resnick M. A., Martin P. The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control. Mol Gen Genet. 1976 Jan 16;143(2):119–129. doi: 10.1007/BF00266917. [DOI] [PubMed] [Google Scholar]
  34. Resnick M. A. The repair of double-strand breaks in DNA; a model involving recombination. J Theor Biol. 1976 Jun;59(1):97–106. doi: 10.1016/s0022-5193(76)80025-2. [DOI] [PubMed] [Google Scholar]
  35. Rose M., Winston F. Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene. Mol Gen Genet. 1984;193(3):557–560. doi: 10.1007/BF00382100. [DOI] [PubMed] [Google Scholar]
  36. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  37. Rothstein R., Helms C., Rosenberg N. Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):1198–1207. doi: 10.1128/mcb.7.3.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ruby S. W., Szostak J. W. Specific Saccharomyces cerevisiae genes are expressed in response to DNA-damaging agents. Mol Cell Biol. 1985 Jan;5(1):75–84. doi: 10.1128/mcb.5.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stahl M. M., Kobayashi I., Stahl F. W., Huntington S. K. Activation of Chi, a recombinator, by the action of an endonuclease at a distant site. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2310–2313. doi: 10.1073/pnas.80.8.2310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Strathern J. N., Klar A. J., Hicks J. B., Abraham J. A., Ivy J. M., Nasmyth K. A., McGill C. Homothallic switching of yeast mating type cassettes is initiated by a double-stranded cut in the MAT locus. Cell. 1982 Nov;31(1):183–192. doi: 10.1016/0092-8674(82)90418-4. [DOI] [PubMed] [Google Scholar]
  41. Struhl K. Effect of deletion and insertion on double-strand-break repair in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):1300–1303. doi: 10.1128/mcb.7.3.1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tatchell K., Nasmyth K. A., Hall B. D., Astell C., Smith M. In vitro mutation analysis of the mating-type locus in yeast. Cell. 1981 Nov;27(1 Pt 2):25–35. doi: 10.1016/0092-8674(81)90357-3. [DOI] [PubMed] [Google Scholar]
  43. Taylor A. F., Schultz D. W., Ponticelli A. S., Smith G. R. RecBC enzyme nicking at Chi sites during DNA unwinding: location and orientation-dependence of the cutting. Cell. 1985 May;41(1):153–163. doi: 10.1016/0092-8674(85)90070-4. [DOI] [PubMed] [Google Scholar]
  44. Weiffenbach B., Haber J. E. Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae. Mol Cell Biol. 1981 Jun;1(6):522–534. doi: 10.1128/mcb.1.6.522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Weiffenbach B., Rogers D. T., Haber J. E., Zoller M., Russell D. W., Smith M. Deletions and single base pair changes in the yeast mating type locus that prevent homothallic mating type conversions. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3401–3405. doi: 10.1073/pnas.80.11.3401. [DOI] [PMC free article] [PubMed] [Google Scholar]

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