In Vitro Reconstitution of BRCA1-BARD1/RAD51-Mediated Homologous DNA Pairing.
Methods Mol Biol
; 2444: 207-225, 2022.
Article
en En
| MEDLINE
| ID: mdl-35290640
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Recombinasa Rad51
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos