In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing

Meiling Wang, Cody M. Rogers, Dauren Alimbetov, Weixing Zhao

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

RAD51-mediated homologous recombination (HR) is a conserved mechanism for the repair of DNA double-strand breaks and the maintenance of DNA replication forks. Several breast and ovarian tumor suppressors, including BRCA1 and BARD1, have been implicated in HR since their discovery in the 1990s. However, a holistic understanding of how they participate in HR has been hampered by the immense challenge of expressing and purifying these large and unstable protein complexes for mechanistic analysis. Recently, we have overcome such a challenge for the BRCA1-BARD1 complex, allowing us to demonstrate its pivotal role in HR via the promotion of RAD51-mediated DNA strand invasion. In this chapter, we describe detailed procedures for the expression and purification of the BRCA1-BARD1 complex and in vitro assays using this tumor suppressor complex to examine its ability to promote RAD51-mediated homologous DNA pairing. This includes two distinct biochemical assays, namely, D-loop formation and synaptic complex assembly. These methods are invaluable for studying the BRCA1-BARD1 complex and its functional interplay with other factors in the HR process.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages207-225
Number of pages19
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2444
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • BRCA1-BARD1
  • D-loop formation
  • Homologous recombination
  • RAD51
  • Synaptic complex assembly

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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