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

Curtis D. Hodge; Ismail H. Ismail; Ross A. Edwards; Greg L. Hura; Andrew T. Xiao; John A. Tainer; Michael J. Hendzel; J. N. Mark Glover

doi:10.1074/jbc.M116.715698

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

From the ^‡Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada,
the ^§Department of Oncology, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada,
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
the ^¶Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt

↵3 To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-2136; Fax: 780-492-0886; E-mail: mark.glover{at}ualberta.ca.

↵1 Both authors contributed equally to this work.

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.

Footnotes

↵2 Supported by a Robert A. Welch Distinguished Chair in Chemistry.
↵* This work was supported in part by Canadian Cancer Society/Canadian Breast Cancer Research Alliance (to J. N. M. G.), Canadian Institutes of Health Research Grants CIHR114975 (to J. N. M. G.) and CIHR119515 (to M. J. H.), National Institutes of Health Grant CA92584 (to J. N. M. G. and J. A. T.), and the Alberta Innovates Health Solutions. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
We dedicate this study in memory of Stephen J. Campbell whose hard work and affable personality made a difference.
The atomic coordinates and structure factors (code 4WHV) have been deposited in the Protein Data Bank (http://wwpdb.org/).
SAXS curves have been deposited to Small Angle Scattering Biological Data Bank (http://www.sasbdb.org/) with the codes: WT RNF8-Ubc13∼Ub: SASDBR3, L451D RNF8-Ubc13∼Ub: SASDBT3, WT RNF8-Ubc13∼Ub/Mms2: SASDBS3, L451D RNF8-Ubc13∼Ub/Mms2: SASDBU3.