SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage
- Yili Yin1,
- Anne Seifert1,
- Joy Shijia Chua1,3,
- Jean-François Maure1,
- Filip Golebiowski1,2 and
- Ronald T. Hay1,4
- 1Wellcome Trust Centre for Gene Regulation and Expression,
- 2Scottish Institute for Cell Signalling, University of Dundee, Dundee DD1 5EH, United Kingdom
Abstract
Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress.
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Footnotes
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↵4 Corresponding author.
E-mail r.t.hay{at}dundee.ac.uk.
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Supplemental material is available for this article.
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Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.189274.112.
- Received February 7, 2012.
- Accepted April 30, 2012.
- Copyright © 2012 by Cold Spring Harbor Laboratory Press
Freely available online through the Genes & Development Open Access option.