SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

Genes Dev. 2012 Jun 1;26(11):1196-208. doi: 10.1101/gad.189274.112.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Cell Cycle Proteins
  • Cell Line, Tumor
  • DNA Breaks, Double-Stranded
  • DNA Damage*
  • DNA Repair*
  • HeLa Cells
  • Homologous Recombination
  • Humans
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Rats
  • Trans-Activators / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • MDC1 protein, human
  • Nuclear Proteins
  • RNF4 protein, human
  • Trans-Activators
  • Transcription Factors
  • Ubiquitin
  • Rnf4 protein, rat
  • Ubiquitin-Protein Ligases