RNF8 regulates assembly of RAD51 at DNA double-strand breaks in the absence of BRCA1 and 53BP1

Cancer Res. 2012 Oct 1;72(19):4974-83. doi: 10.1158/0008-5472.CAN-12-1057. Epub 2012 Aug 3.

Abstract

The tumor suppressor protein BRCA1 localizes to sites of DNA double-strand breaks (DSB), promoting repair by homologous recombination through the recruitment of DNA damage repair proteins. In normal cells, homologous recombination largely depends on BRCA1. However, assembly of the pivotal homologous recombination regulator RAD51 can occur independently of BRCA1 in the absence of 53BP1, another DNA damage response protein. How this assembly process proceeds is unclear, but important to understand in tumor cell settings where BRCA1 is disabled. Here we report that RNF8 regulates BRCA1-independent homologous recombination in 53BP1-depleted cells. RNF8 depletion suppressed the recruitment of RAD51 to DSB sites without affecting assembly or phosphorylation of the replication protein RPA in neocarzinostatin-treated or X-ray-irradiated BRCA1/53BP1-depleted cells. Furthermore, RNF8/BRCA1/53BP1-depleted cells exhibited less efficient homologous recombination than BRCA1/53BP1-depleted cells. Intriguingly, neither RNF8 nor its relative RNF168 were required for RAD51 assembly at DSB sites in 53BP1-expressing cells. Moreover, RNF8-independent RAD51 assembly was found to be regulated by BRCA1. Together, our findings indicate a tripartite regulation of homologous recombination by RNF8, BRCA1, and 53BP1. In addition, our results predict that RNF8 inhibition may be a useful treatment of BRCA1-mutated/53BP1(low) cancers, which are considered resistant to treatment by PARP1 inhibitors and of marked current clinical interest.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • BRCA1 Protein / genetics
  • BRCA1 Protein / metabolism*
  • Blotting, Western
  • Cells, Cultured
  • Chromatin / genetics
  • Chromatin / metabolism
  • DNA Breaks, Double-Stranded
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Embryo, Mammalian / cytology
  • Fibroblasts / metabolism
  • HCT116 Cells
  • HEK293 Cells
  • HeLa Cells
  • Homologous Recombination
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mice
  • Mice, Knockout
  • Microscopy, Fluorescence
  • RNA Interference
  • Rad51 Recombinase / metabolism*
  • Tumor Suppressor p53-Binding Protein 1
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • BRCA1 Protein
  • Chromatin
  • DNA-Binding Proteins
  • Intracellular Signaling Peptides and Proteins
  • RNF8 protein, human
  • TP53BP1 protein, human
  • Tumor Suppressor p53-Binding Protein 1
  • UBE2N protein, human
  • Ubiquitin-Conjugating Enzymes
  • RNF168 protein, human
  • Ubiquitin-Protein Ligases
  • RAD51 protein, human
  • Rad51 Recombinase