RNF8-dependent and RNF8-independent Regulation of 53BP1 in Response to DNA Damage

Abstract

The DNA damage surveillance network orchestrates cellular responses to DNA damage through the recruitment of DNA damage-signaling molecules to DNA damage sites and the concomitant activation of protein phosphorylation cascades controlled by the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases. Activation of ATM/ATR triggers cell cycle checkpoint activation and adaptive responses to DNA damage. Recent studies suggest that protein ubiquitylation or degradation plays an important role in the DNA damage response. In this study, we examined the potential role of the proteasome in checkpoint activation and ATM/ATR signaling in response to UV light-induced DNA damage. HeLa cells treated with the proteasome inhibitor MG-132 showed delayed phosphorylation of ATM substrates in response to UV light. UV light-induced phosphorylation of 53BP1, as well as its recruitment to DNA damage foci, was strongly suppressed by proteasome inhibition, whereas the recruitment of upstream regulators of 53BP1, including MDC1 and H2AX, was unaffected. The ubiquitin-protein isopeptide ligase RNF8 was critical for 53BP1 focus targeting and phosphorylation in ionizing radiation-damaged cells, whereas UV light-induced 53BP1 phosphorylation and targeting exhibited partial dependence on RNF8 and the ubiquitin-conjugating enzyme UBC13. Suppression of RNF8 or UBC13 also led to subtle defects in UV light-induced G 2 /M checkpoint activation. These findings are consistent with a model in which RNF8 ubiquitylation pathways are essential for 53BP1 regulation in response to ionizing radiation, whereas RNF8-independent pathways contribute to 53BP1 targeting and phosphorylation in response to UV light and potentially other forms of DNA replication stress.DNA-damaging stimuli elicit highly conserved responses in eukaryotic cells that are required for the maintenance of genomic integrity and organismal viability. Protein kinase cascades are central to the DNA damage-signaling paradigm, and members of the phosphoinositide 3-kinase-related kinase gene superfamily play particularly important roles as apical DNA damage response regulators. Two functionally related members of the phosphoinositide 3-kinase-related kinase family, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) kinases, initiate overlapping signaling pathways in response to distinct types of genetic lesions. ATM is principally activated by ionizing radiation (IR) 2 and other agents that induce DNA double-strand breaks, whereas ATR responds to pauses in DNA replication that transiently expose single-strand DNA (1). Once activated, ATM and ATR phosphorylate and activate the CHK2 and CHK1 protein kinases, respectively, which in turn promote cell cycle checkpoint arrest through inhibition of CDC25 family phosphatases (2-4). ATM and ATR phosphorylate numerous other substrates, including p53 and BRCA1, which participate in DNA metabolism. The regulatory reach of ATM and ATR extends to many other physiologic processes, and recent studies suggest that the true numb...

Transcription-dependent Activation of Ataxia Telangiectasia Mutated Prevents DNA-dependent Protein Kinase-mediated Cell Death in Response to Topoisomerase I Poison

“…SV40-transformed GM00637H (ATMϩ), GM05849C (ATMϪ), (obtained from Coriell Cell Repositories), and hTERT-immortalized SuSa/Tn (ATMϩ), AT1OS/Tn (ATMϪ) (kindly provided by Dr. K Ishizaki) (8) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Cells were UV-and IR-irradiated as reported (9). CPT, VP-16, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB), KU-55933, NU7026, hydroxyurea (HU), and thymidine were purchased from Sigma.…”

“…Western Blotting-Cell lysis, SDS-PAGE, and gel transfer were performed as described (9). After incubation with secondary antibody, the membranes were visualized using SuperSignal chemiluminescent substrate (Pierce).…”

“…Cell preparation for staining of 53BP1, ␥H2AX and incorporated BrdUrd was performed as described (9). For RPA2 and Rad51 staining, cells were preextracted with phosphate-buffered saline containing 0.1% Triton X-100.…”

Chromatin dynamics coupled to DNA repair

“…Nevertheless, RAP80 does not contribute to 53BP1 translocation and it has been proposed that H2A and H2AX ubiquitination may promote changes in chromatin conformation which may increase the exposure of methylated histone, the targets of 53BP1 in the chromatin. 68 …”

53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope

“…So far, 53BP1 is considered as a scaffold protein that is devoid of any enzymatic activity, and hence is unlikely to constitute a pharmacological target. However, 53BP1 is affected in its activity by enzymes, such as the ubiquitin ligase RNF168, 35 and it will be important to study whether the inhibition of such upstream factors will suppress syncytial apoptosis (as this has been found for ATM inhibition) or rather exacerbate synctial apoptosis. On theoretical grounds, agents that induce the selective death of HIV-1-elicited syncytia might lead to the elimination of viral reservoirs and hence constitute a complement to current antiretroviral therapies.…”