ruitment of Phosphorylated NPM1 to Sites of DNA

ownloade tein accumulation at DNA double-strand breaks (DSB) is essential for genome stability; however, the nisms governing these events are not fully understood. Here, we report a new role for the nucleophosrotein NPM1 in these mechanisms. Thr199-phosphorylated NPM1 (pT199-NPM1) is recruited to nuclear amage foci induced by ionizing radiation (IR). Foci formation is impaired by depletion of the E3 ubiligases RNF8 and RNF168 or the E2 Ubc13, and pT199-NPM1 binds to Lys63-linked ubiquitin polymers o. Thus, phosphorylated NPM1 may interact with RNF8-dependent ubiquitin conjugates at sites of DNA e. The interaction was found to rely on T199 phosphorylation, an acidic tract, and an adjacent ubiquitincting motif–like domain. Depletion of the breast cancer suppressor BRCA1 or its partner, RAP80, ced IR-induced NPM1 foci and prolonged persistence of the foci, possibly implicating BRCA1 in -NPM1 action and dynamics. Replacement of endogenous NPM1 with its nonphosphorylable mutant prolonged persistence of IR-induced RAD51 foci accompanied by unrepaired DNA damage. T199A Collectively, our findings suggest that phosphorylated NPM1 is a novel component in DSB repair that is recruited by ubiquitin conjugates downstream of RNF8 and RNF168. Cancer Res; 70(17); OF1–11. ©2010 AACR.