Single-molecule studies of yeast Rad51 paralogs

Upasana Roy, Youngho Kwon, Patrick Sung, Eric C. Greene

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Homologous recombination (HR) is a conserved mechanism essential for the accurate repair of DNA double stranded breaks and the exchange of genetic information during meiosis. The key steps in HR are carried out by the RecA/Rad51 class of recombinases, which form a helical filament on single-stranded DNA (ssDNA) and catalyze homology search and strand exchange with a complementary duplex DNA target. In eukaryotes, assembly of the Rad51–ssDNA filament requires regulatory factors called mediators, including Rad51 paralogs. A mechanistic understanding of the role of Rad51 paralogs in HR has been hampered by the transient and diverse nature of intermediates formed with the Rad51–ssDNA filament, which cannot be resolved by traditional ensemble methods. The biochemical characterization of Rad51 paralogs, including the S. cerevisiae complex Rad55–Rad57 has also been limited by their propensity to aggregate. Here we describe the preparation of monodisperse GFP-tagged Rad55–Rad57 complex and the methodology for its analysis in our single-molecule DNA curtain assay.

Original languageEnglish (US)
Title of host publicationThe DNA Replication-Repair Interface
EditorsBrandt F. Eichman
PublisherAcademic Press Inc.
Pages343-362
Number of pages20
ISBN (Print)9780323907330
DOIs
StatePublished - Jan 2021

Publication series

NameMethods in Enzymology
Volume661
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • DNA curtain
  • Homologous recombination
  • Rad51 paralogs
  • Rad55–Rad57
  • Single-molecule

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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