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Genetic and Molecular Analysis of Mitotic Recombination in Saccharomyces cerevisiae

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DNA Recombination

Part of the book series: Methods in Molecular Biology ((MIMB,volume 745))

Abstract

Many systems have been developed for the study of mitotic homologous recombination (HR) in the yeast Saccharomyces cerevisiae at both genetic and molecular levels. Such systems are of great use for the analysis of different features of HR as well as of the effect of mutations, transcription, etc., on HR. Here we describe a selection of plasmid- and chromosome-borne DNA repeat assays, as well as plasmid–chromosome recombination systems, which are useful for the analysis of spontaneous and DSB-induced recombination. They can easily be used in diploid and, most importantly, in haploid yeast cells, which is a great advantage to analyze the effect of recessive mutations on HR. Such systems were designed for the analysis of a number of different HR features, which include the frequency and length of the gene conversion events, the frequency of reciprocal exchanges, the proportion of gene conversion versus reciprocal exchange, or the molecular analysis of sister chromatid exchange.

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References

  1. McClintock, B. (1938) The production of homozygous deficient tissues with mutant characteristics by means of the aberrant mitotic behavior of ring-shaped chromosomes. Genetics 23, 315–376.

    PubMed  CAS  Google Scholar 

  2. Gonzalez-Barrera, S., Cortes-Ledesma, F., Wellinger, R.E., and Aguilera, A. (2003) Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast. Mol Cell 11, 1661–1671.

    Article  PubMed  CAS  Google Scholar 

  3. Kadyk, L.C., and Hartwell, L.H. (1992) Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cerevisiae. Genetics 132, 387–402.

    PubMed  CAS  Google Scholar 

  4. Pardo, B., Gomez-Gonzalez, B., and Aguilera, A. (2009) DNA repair in mammalian cells: DNA double-strand break repair: how to fix a broken relationship. Cell Mol Life Sci 66, 1039–1056.

    Article  PubMed  CAS  Google Scholar 

  5. Barbera, M.A., and Petes, T.D. (2006) Selection and analysis of spontaneous reciprocal mitotic cross-overs in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 103, 12819–12824.

    Article  PubMed  CAS  Google Scholar 

  6. Jackson, J.A., and Fink, G.R. (1981) Gene conversion between duplicated genetic elements in yeast. Nature 292, 306–311.

    Article  PubMed  CAS  Google Scholar 

  7. Klein, H.L., and Petes, T.D. (1981) Intrachromosomal gene conversion in yeast. Nature 289, 144–148.

    Article  PubMed  CAS  Google Scholar 

  8. Prado, F., Cortes-Ledesma, F., Huertas, P., and Aguilera, A. (2003) Mitotic recombination in Saccharomyces cerevisiae. Curr Genet 42, 185–198.

    PubMed  CAS  Google Scholar 

  9. Cortes-Ledesma, F., Prado, F., and Aguilera, A. (2007) Molecular genetics of recombination. Top Curr Genet 17, 221–250.

    Article  CAS  Google Scholar 

  10. Aguilera, A., and Klein, H.L. (1988) Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics 119, 779–790.

    PubMed  CAS  Google Scholar 

  11. Judd, S.R., and Petes, T.D. (1988) Physical lengths of meiotic and mitotic gene conversion tracts in Saccharomyces cerevisiae. Genetics 118, 401–410.

    PubMed  CAS  Google Scholar 

  12. Wallis, J.W., Chrebet, G., Brodsky, G., Rolfe, M., and Rothstein, R. (1989) A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell 58, 409–419.

    Article  PubMed  CAS  Google Scholar 

  13. Rudin, N., Sugarman, E., and Haber, J.E. (1989) Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae. Genetics 122, 519–534.

    PubMed  CAS  Google Scholar 

  14. Fasullo, M., Giallanza, P., Dong, Z., Cera, C., and Bennett, T. (2001) Saccharomyces cerevisiae rad51 mutants are defective in DNA damage-associated sister chromatid exchanges but exhibit increased rates of homology-directed translocations. Genetics 158, 959–972.

    PubMed  CAS  Google Scholar 

  15. Saxe, D., Datta, A., and Jinks-Robertson, S. (2000) Stimulation of mitotic recombination events by high levels of RNA polymerase II transcription in yeast. Mol Cell Biol 20, 5404–5414.

    Article  PubMed  CAS  Google Scholar 

  16. Voelkel-Meiman, K., Keil, R.L., and Roeder, G.S. (1987) Recombination-stimulating sequences in yeast ribosomal DNA correspond to sequences regulating transcription by RNA polymerase I. Cell 48, 1071–1079.

    Article  PubMed  CAS  Google Scholar 

  17. Rattray, A.J., and Symington, L.S. (1994) Use of a chromosomal inverted repeat to demonstrate that the RAD51 and RAD52 genes of Saccharomyces cerevisiae have different roles in mitotic recombination. Genetics 138, 587–595.

    PubMed  CAS  Google Scholar 

  18. Ahn, B.Y., and Livingston, D.M. (1986) Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system. Mol Cell Biol 6, 3685–3693.

    PubMed  CAS  Google Scholar 

  19. Kupiec, M., and Petes, T.D. (1988) Meiotic recombination between repeated transposable elements in Saccharomyces cerevisiae. Mol Cell Biol 8, 2942–2954.

    PubMed  CAS  Google Scholar 

  20. Aguilera, A., and Klein, H.L. (1989) Genetic and molecular analysis of recombination events in Saccharomyces cerevisiae occurring in the presence of the hyper-recombination mutation hpr1. Genetics 122, 503–517.

    PubMed  CAS  Google Scholar 

  21. Prado, F., and Aguilera, A. (1995) Role of reciprocal exchange, one-ended invasion crossover and single-strand annealing on inverted and direct repeat recombination in yeast: different requirements for the RAD1, RAD10, and RAD52 genes. Genetics 139, 109–123.

    PubMed  CAS  Google Scholar 

  22. Chavez, S., and Aguilera, A. (1997) The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability. Genes Dev 11, 3459–3470.

    Article  PubMed  CAS  Google Scholar 

  23. Prado, F., Piruat, J.I., and Aguilera, A. (1997) Recombination between DNA repeats in yeast hpr1delta cells is linked to transcription elongation. EMBO J 16, 2826–2835.

    Article  PubMed  CAS  Google Scholar 

  24. Garcia-Rubio, M., Huertas, P., Gonzalez-Barrera, S., and Aguilera, A. (2003) Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae. New insights into transcription-associated recombination. Genetics 165, 457–466.

    PubMed  CAS  Google Scholar 

  25. Gomez-Gonzalez, B., and Aguilera, A. (2007) Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex. Proc Natl Acad Sci USA 104, 8409–8414.

    Article  PubMed  CAS  Google Scholar 

  26. Aguilera, A., and Klein, H.L. (1989) Yeast intrachromosomal recombination: long gene conversion tracts are preferentially associated with reciprocal exchange and require the RAD1 and RAD3 gene products. Genetics 123, 683–694.

    PubMed  CAS  Google Scholar 

  27. Malagon, F., and Aguilera, A. (2001) Yeast spt6-140 mutation, affecting chromatin and transcription, preferentially increases recombination in which Rad51p-mediated strand exchange is dispensable. Genetics 158, 597–611.

    PubMed  CAS  Google Scholar 

  28. Gonzalez-Barrera, S., Garcia-Rubio, M., and Aguilera, A. (2002) Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae. Genetics 162, 603–614.

    PubMed  CAS  Google Scholar 

  29. Fasullo, M.T., and Davis, R.W. (1987) Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo. Proc Natl Acad Sci USA 84, 6215–6219.

    Article  PubMed  CAS  Google Scholar 

  30. Fasullo, M., Bennett, T., and Dave, P. (1999) Expression of Saccharomyces cerevisiae MATa and MAT alpha enhances the HO endonuclease-stimulation of chromosomal rearrangements directed by his3 recombinational substrates. Mutat Res 433, 33–44.

    PubMed  CAS  Google Scholar 

  31. Cortes-Ledesma, F., and Aguilera, A. (2006) Double-strand breaks arising by replication through a nick are repaired by cohesin-dependent sister-chromatid exchange. EMBO Rep 7, 919–926.

    Article  PubMed  CAS  Google Scholar 

  32. Holmes, K.L., Otten, G., and Yokoyama, W.M. (2002) Flow cytometry analysis using the Becton Dickinson FACS Calibur. Curr Protoc Immunol 5, 54.

    Google Scholar 

  33. Lea, D.E., and Coulson, C.A. (1948) The distribution of the number of mutants in bacterial population. J Genet 49, 264–284.

    Article  Google Scholar 

  34. Schmidt, K.H., Pennaneach, V., Putnam, C.D., and Kolodner, R.D. (2006) Analysis of gross-chromosomal rearrangements in Saccharomyces cerevisiae. Meth Enzymol 409, 462–476.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank H. Gaillard and M. Moriel-Carretero for reading the manuscript and D. Haun for style supervision. We apologize for not citing our colleague’s work due to space limitation. Research in the A. A. laboratory was funded by research grants from the Spanish Ministry of Science and Innovation and the Junta de Andalucía.

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Authors and Affiliations

  1. Centro Andaluz de Biología Molecular y Medicina Regenerativa, Universidad de Sevilla-CSIC, Sevilla, Spain

    Belén Gómez-González, José F. Ruiz & Andrés Aguilera

Authors
  1. Belén Gómez-González
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  2. José F. Ruiz
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  3. Andrés Aguilera
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Corresponding author

Correspondence to Belén Gómez-González .

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Editors and Affiliations

  1. MRC Genome Damage and Stability Centre, University of Sussex, Science Park Road, Brighton, BN1 9RQ, East Sussex, United Kingdom

    Hideo Tsubouchi

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Gómez-González, B., Ruiz, J.F., Aguilera, A. (2011). Genetic and Molecular Analysis of Mitotic Recombination in Saccharomyces cerevisiae . In: Tsubouchi, H. (eds) DNA Recombination. Methods in Molecular Biology, vol 745. Humana Press. https://doi.org/10.1007/978-1-61779-129-1_10

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  • DOI: https://doi.org/10.1007/978-1-61779-129-1_10

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-128-4

  • Online ISBN: 978-1-61779-129-1

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