RNF8 regulates assembly of RAD51 at DNA double-strand breaks in the absence of BRCA1 and 53BP1.

The tumor suppressor protein BRCA1 localizes to sites of DNA double-strand breaks (DSB), promoting repair by homologous recombination through the recruitment of DNA damage repair proteins. In normal cells, homologous recombination largely depends on BRCA1. However, assembly of the pivotal homologous recombination regulator RAD51 can occur independently of BRCA1 in the absence of 53BP1, another DNA damage response protein. How this assembly process proceeds is unclear, but important to understand in tumor cell settings where BRCA1 is disabled. Here we report that RNF8 regulates BRCA1-independent homologous recombination in 53BP1-depleted cells. RNF8 depletion suppressed the recruitment of RAD51 to DSB sites without affecting assembly or phosphorylation of the replication protein RPA in neocarzinostatin-treated or X-ray-irradiated BRCA1/53BP1-depleted cells. Furthermore, RNF8/BRCA1/53BP1-depleted cells exhibited less efficient homologous recombination than BRCA1/53BP1-depleted cells. Intriguingly, neither RNF8 nor its relative RNF168 were required for RAD51 assembly at DSB sites in 53BP1-expressing cells. Moreover, RNF8-independent RAD51 assembly was found to be regulated by BRCA1. Together, our findings indicate a tripartite regulation of homologous recombination by RNF8, BRCA1, and 53BP1. In addition, our results predict that RNF8 inhibition may be a useful treatment of BRCA1-mutated/53BP1(low) cancers, which are considered resistant to treatment by PARP1 inhibitors and of marked current clinical interest.

Nakada S ，Yonamine RM ，Matsuo K 《-》

Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks.

Poulsen M ，Lukas C ，Lukas J ，Bekker-Jensen S ，Mailand N ... -  《-》

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Hodge CD ，Ismail IH ，Edwards RA ，Hura GL ，Xiao AT ，Tainer JA ，Hendzel MJ ，Glover JN ... -  《-》

RING finger nuclear factor RNF168 is important for defects in homologous recombination caused by loss of the breast cancer susceptibility factor BRCA1.

Muñoz MC ，Laulier C ，Gunn A ，Cheng A ，Robbiani DF ，Nussenzweig A ，Stark JM ... -  《-》

Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.

Kolas NK ，Chapman JR ，Nakada S ，Ylanko J ，Chahwan R ，Sweeney FD ，Panier S ，Mendez M ，Wildenhain J ，Thomson TM ，Pelletier L ，Jackson SP ，Durocher D ... -  《-》