Protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation

STAR Protoc. 2024 Sep 20;5(3):103294. doi: 10.1016/j.xpro.2024.103294. Epub 2024 Sep 6.

Abstract

The tumor suppressor breast cancer 1 (BRCA1) complexed with BRCA1-associated RING domain 1 (BARD1), a RING-type E3 ligase, facilitates the attachment of ubiquitin onto the substrate protein. Here, we present a protocol for evaluating the E3 ligase activity of BRCA1-BARD1 and its variants by nucleosomal histone ubiquitylation. We describe steps for isolating 147 bp Widom 601 DNA and assembling nucleosome core particles (NCPs). We then detail procedures for the in vitro ubiquitylation of nucleosome histone H2A by BRCA1-BARD1 and its variants. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Keywords: Cancer; Cell culture; Protein expression and purification.

MeSH terms

  • BRCA1 Protein* / genetics
  • BRCA1 Protein* / metabolism
  • Histones* / genetics
  • Histones* / metabolism
  • Humans
  • Nucleosomes* / genetics
  • Nucleosomes* / metabolism
  • Tumor Suppressor Proteins* / genetics
  • Tumor Suppressor Proteins* / metabolism
  • Ubiquitin-Protein Ligases* / genetics
  • Ubiquitin-Protein Ligases* / metabolism
  • Ubiquitination*

Substances

  • Ubiquitin-Protein Ligases
  • Nucleosomes
  • BRCA1 Protein
  • Histones
  • BRCA1 protein, human
  • BARD1 protein, human
  • Tumor Suppressor Proteins