Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

Nucleic Acids Res. 2013 Dec;41(22):10298-311. doi: 10.1093/nar/gkt802. Epub 2013 Sep 5.

Abstract

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using α-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1's BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from non-homologous end-joining to homologous recombination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • BRCA1 Protein / chemistry
  • BRCA1 Protein / metabolism*
  • Carrier Proteins / metabolism*
  • Carrier Proteins / physiology
  • Cells, Cultured
  • DNA-Binding Proteins
  • Endodeoxyribonucleases
  • G2 Phase / genetics
  • Histone Chaperones
  • Histones / analysis
  • Homologous Recombination*
  • Humans
  • Intracellular Signaling Peptides and Proteins / analysis
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Mice
  • Nuclear Proteins / metabolism*
  • Nuclear Proteins / physiology
  • Proteasome Endopeptidase Complex / metabolism*
  • Proteasome Endopeptidase Complex / physiology
  • Protein Structure, Tertiary
  • Trans-Activators / metabolism*
  • Trans-Activators / physiology
  • Tumor Suppressor p53-Binding Protein 1
  • Ubiquitin / analysis
  • Ubiquitin / metabolism

Substances

  • BRCA1 Protein
  • Carrier Proteins
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histone Chaperones
  • Histones
  • Intracellular Signaling Peptides and Proteins
  • Nuclear Proteins
  • PSMD14 protein, human
  • TP53BP1 protein, human
  • Trans-Activators
  • Tumor Suppressor p53-Binding Protein 1
  • UIMC1 protein, human
  • Ubiquitin
  • Endodeoxyribonucleases
  • RBBP8 protein, human
  • Proteasome Endopeptidase Complex