Reconstituted System for the Examination of Repair DNA Synthesis in Homologous Recombination

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations

Abstract

In homologous recombination (HR), DNA polymerase δ-mediated DNA synthesis occurs within the displacement loop (D-loop) that is made by the recombinase Rad51 in conjunction with accessory factors. We describe in this chapter the reconstitution of the D-loop and repair DNA synthesis reactions using purified Saccharomyces cerevisiae HR (Rad51, RPA, and Rad54) and DNA replication (PCNA, RFC, and DNA polymerase δ) proteins and document the role of the Pif1 helicase in DNA synthesis via a migrating DNA bubble intermediate. These reconstituted systems are particularly valuable for understanding the conserved mechanism of repair DNA synthesis dependent on DNA polymerase δ and its cognate helicase in eukaryotic organisms.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages307-325
Number of pages19
DOIs
StatePublished - 2017
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume591
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • BIR
  • D-loop
  • DNA polymerase δ
  • DNA synthesis
  • Homologous recombination
  • Pif1

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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