[PMC free article] [PubMed] [Google Scholar] 28. a novel post-translational modification of 53BP1 by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 polyubiquitination and degradation. In response to DNA damage, ADP-ribosylated 53BP1 increased significantly, resulting in its ubiquitination and degradation. These data suggest that NUDT16 plays a major role in controlling 53BP1 levels under both normal growth conditions and during DNA damage. Notably, overexpression of a NUDT16 catalytically inactive mutant blocked 53BP1 localization to double-strand breaks because: 1) the mutant binding to TIRR increased after IR; 2) the mutant enhanced 53BP1 Tudor domain binding to TIRR, and 3) the mutant impaired the interaction of 53BP1 Tudor domain with H4K20me2. Moreover, NUDT16s catalytic hydrolase activity was required for 53BP1 de-ADP-ribosylation, 53BP1 protein stability, and its function in cell survival. In summary, we demonstrate that NUDT16 regulates 53BP1 stability and 53BP1 recruitment at double-strand breaks, providing yet another mechanism of 53BP1 regulation. INTRODUCTION DNA double-strand breaks (DSBs) are the most consequential type of DNA damage because, if unrepaired, they lead to genetic instability and are implicated in many human diseases. Two distinct pathways are involved in the repair of DNA DSBs: non-homologous DNA end- joining (NHEJ), which rejoins the broken ends without the use of extensive homology; and homologous recombination (HR), which requires a homology template from an undamaged sister chromatid chromosome. A deep understanding of DNA repair pathway choice not only is crucial to our understanding of cancer, but also provides new targets for cancer therapies. The protein 53BP1 has attracted particular Bavisant attention in DNA repair because of its role in the choice between NHEJ and HR and its relevance to PARP inhibitor (PARPi) treatment of BRCA1-mutant cancers. 53BP1 contains a highly phosphorylated N-terminal region, a Tudor domain in the middle, and two BRCT domains at its C-terminus. The formation of 53BP1 foci at DNA damage sites requires binding of H4K20me2 via its Tudor Cdx1 domain (1). In support of the roles that histone methylation plays in 53BP1 localization, one of the histone methyltransferases, MMSET, has been implicated in 53BP1 recruitment at sites of DNA damage (2). The IRIF (ionizing radiation-induced foci) region of 53BP1 also contains a ubiquitination-dependent recruitment motif. Recent studies suggest that accumulation of 53BP1 at DNA damage sites requires both domains plus histone ubiquitination at H2AX K13/K15 sites (3), which is mediated by RNF8/RNF168 in the H2AX-dependent pathway (4C9). 53BP1 is generally hypothesized to be an adaptor protein that does not act directly in DNA repair. Instead, it recruits other proteins to the sites of DNA breaks to facilitate NHEJ repair, which then compete with BRCA1-dependent HR-mediated DNA repair. In support of this hypothesis, several groups demonstrated that RIF1 and PTIP are key factors that act downstream of 53BP1 and counteract BRCA1s functions in DNA repair (10C15). However, RIF1 and PTIP have no known enzymatic activity in DNA repair and rather, may serve as a scaffold for the recruitment of other DSB-processing enzymes in this pathway. Recently, we and others reported that downstream of 53BP1, Artemis and MDA2L2 are crucial in coordinating pathologic DSB repair pathway choices in BRCA1-deficient cells. Loss of Artemis or MAD2L2 restores PARPi resistance in BRCA1-deficient cells (16C18). More recently, the Shieldin complex was found to act as the downstream effector of 53BP1/RIF1/MAD2L2 to restrict DSB resection and counteract HR (19C24). Thus, 53BP1 controls two downstream pathways, one mediated by PTIP and Artemis and the other Bavisant by RIF1 and MAD2L2/Shieldin, to promote NHEJ and suppress HR repair. Although 53BP1 has been extensively investigated for its downstream pathways and DNA repair pathway choice, little is known about upstream regulation of 53BP1 function in DNA repair. In particular, it remains unclear how post-translational modification of 53BP1 affects DSB repair. We and others recently showed that TIRR (Tudor interacting repair regulator) directly binds to the 53BP1 Tudor domain, masks its binding surface for H4K20me2, and regulates 53BP1 functions (25C29). Given that the TIRR homolog, Nudix enzyme NUDT16, Bavisant has been shown to have hydrolase activities that remove protein ADP-ribosylation (30), we investigated whether NUDT16 regulates 53BP1 stability and we are now reporting that by hydrolyzing PAR chains on 53BP1, NUDT16 indeed regulates 53BP1 stability. MATERIALS AND METHODS Cell culture and plasmids HEK293T, MCF10A, and MDA-MB-231 cells were purchased from the ATCC and cultured under conditions specified by the ATCC. All cell lines were cultured within.