Journal article
RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling
F Mattiroli, JHA Vissers, WJ Van Dijk, P Ikpa, E Citterio, W Vermeulen, JA Marteijn, TK Sixma
Cell | CELL PRESS | Published : 2012
Abstract
Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find..
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Awarded by European Research Council
Funding Acknowledgements
We thank A. Perrakis, P. Rucktooa, and R. Joosten for crystallography support, J. Smit for contribution to E1 and E2 purifications, M. Uckelmann for contribution to in vitro specificity, D. Svergun and F. Groothuizen for assistance in SAXS measurements and analysis, J. Lukas, R. Klevit, L. Penengo, K. Luger, M. Peuscher, and J. Jacobs for plasmids, J. van der Knaap for providing Drosophila oligonucleosomes enriched in ubiquitinated H2B, G. Smeenk for help with gH2AX IPs, R. Hibbert for PCNA and Rad6 samples, J. Chen for anti-RAP80 antibodies, A. Nussenzweig for H2ax<SUP>-/-</SUP> MEFs, Genentech for K63-specific antibodies, W.A. van Cappellen of the Optical Imaging Centre, ErasmusMC, for help with the quantification of foci, and T. Brummelkamp, F. van Leeuwen, colleagues and members of the Sixma laboratory for critical reading of the manuscript. These studies were funded by the ERC Advanced grant, EU project Ubiregulators, KWF 2006-3476 to T.K.S., Dutch Organization for Scientific Research ZonMW Veni 917-96-120 to J.A.M., NWO Vidi 864.08.011 to E.C., and KWF 2007-3877. F.M. designed and performed all experiments and wrote the manuscript. J.H.A.V. contributed to cell-based experiments and discussions. J.A.M., W.V., and E.C. supervised immunofluorescence experiments and imaging. J.A.M. performed quantifications of immunofluorescence data. W.J.vD. assisted in purification and western blots. P.I. cloned and purified the RNF8 RING domain. T.K.S. designed and supervised experiments and wrote the manuscript. All authors critically read the paper.