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J Biol Chem. 2016 Apr 29;291(18):9396-410. doi: 10.1074/jbc.M116.715698. Epub 2016 Feb 22.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.

Author information

1
From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
2
the Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada, the Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt.
3
the Lawrence Berkeley National Laboratory, Berkeley, California 94704.
4
the Lawrence Berkeley National Laboratory, Berkeley, California 94704, the Department of Molecular and Cellular Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, and.
5
the Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada.
6
From the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, mark.glover@ualberta.ca.

Abstract

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

KEYWORDS:

53BP1; BRCA1; DNA damage response; E3 ubiquitin-protein ligase RNF8 (RNF8); RNF168; Ubc13; cell biology; ubiquitylation (ubiquitination); x-ray crystallography; x-ray scattering

PMID:
26903517
PMCID:
PMC4850281
DOI:
10.1074/jbc.M116.715698
[Indexed for MEDLINE]
Free PMC Article

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