SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage.
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.
Wellcome Trust Centre for Gene Regulation and Expression, Dundee, United Kingdom.
Yili
Y
Yin
Anne
A
Seifert
Joy Shijia
JS
Chua
Jean-François
JF
Maure
Filip
F
Golebiowski
Ronald T
RT
Hay
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004260:DNA Repair; D006367:HeLa Cells; D059765:Homologous Recombination; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; C114070:RNF4 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025801:Ubiquitin; C486411:Rnf4 protein, rat; D044767:Ubiquitin-Protein Ligases