RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins.
Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.
Mailand
Niels
N
Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
Bekker-Jensen
Simon
S
Faustrup
Helene
H
Melander
Fredrik
F
Bartek
Jiri
J
Lukas
Claudia
C
Lukas
Jiri
J
D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein / Q000378:metabolism; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002470:Cell Survival; D002843:Chromatin / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000071857:Tumor Suppressor p53-Binding Protein 1; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; C486521:RNF8 protein, human; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; D044767:Ubiquitin-Protein Ligases; D045643:DNA Repair Enzymes