Monitoring Recombination During Meiosis in Budding Yeast

Shannon Owens, Shangming Tang, Neil Hunter

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages275-307
Number of pages33
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Enzymology
Volume601
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Crossover
  • Double-Holliday junction
  • Homologous recombination
  • Joint molecule
  • Meiosis
  • Noncrossover
  • Psoralen
  • Single-end invasion
  • Southern hybridization
  • Two-dimensional (2D) gel electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Owens, S., Tang, S., & Hunter, N. (2018). Monitoring Recombination During Meiosis in Budding Yeast. In Methods in Enzymology (pp. 275-307). (Methods in Enzymology; Vol. 601). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2017.12.005