RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks

Abstract

Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.ubiquitin | G2/M checkpoint | HR repair pathways | NHEJ repair pathway T he DNA-damage response (DDR) is critical for genomic integrity (1) and is regulated by posttranslational modifications (PTMs) such as ubiquitylation of histones by the E3 ligases RING finger 8 (RNF8) and RING finger 168 (RNF168). Other PTMs important for DDR include dimethylation of histone H4 (H4K20me2), which allows p53-binding protein 1 (53BP1), a key mediator of DDR, to interact with chromatin.Mutations of RNF168 have been associated with the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome, (2-4). RNF168 has an N-terminal RING finger domain, three ubiquitin (Ub)-binding domains (UBDs); two motif interacting with Ub (MIU) domains; and one Ub interacting motif (UIM)-and MIU-related (UMI) UBD (3,5,6). Current data support RNF168 function in DNA double-strand break (DSB) signaling downstream of H2A.X, mediator of DNA damage checkpoint 1 (MDC1), and RNF8 and indicate its requirement for 53BP1 recruitment to DSB sites (3, 5). Through its UBDs, RNF168 recognizes RNF8 ubiquitylated non-nucleosomal protein(s) at DSB-flanking sites, leading to its recruitment at these sites of DNA damage (7). With the Ubconjugating enzyme UBC13, RNF168 initiates ubiquitylation of lysine (K) 13 or 15 of histones H2A and H2A.X, leading to the recruitment of DDR proteins, including 53BP1, to DSBs. Although H2A.X, MDC1, and RNF8 are important for the retention of 53BP1 at these DSBs, its initial and transient recruitment to DNA breaks still occurs in their absence (8-11). In contrast, deficiency of Rnf168 in mouse embryonic fibroblasts (MEFs) completely abolishes 53bp1 recruitment to DSB sites (12). Similar to 53bp1 −/− mice, but in contrast to H2a.x −/− mice (13), young Rnf168 −/− males are ferti...

INT6/EIF3E Controls the RNF8-Dependent Ubiquitylation Pathway and Facilitates DNA Double-Strand Break Repair in Human Cells

“…S7B). Interestingly, a study showed that RNF168, but not RNF8, interacts with 53BP1 before their relocalization at DSB and RNF168 catalyzes Lys63-linked ubiquitylation of 53BP1, which is required for the initial recruitment of 53BP1 to DSB sites (34). Our data showing that RNF168 and 53BP1, but not RNF8 or BRCA1, were normally enriched at DSBs following INT6 silencing are in agreement with this work.…”

“…Insights provided by previous studies may help in solving this apparent paradox. Bohgaki et al (2013) showed that RNF168 interacts with 53BP1 before their assembly at DSB and RNF168 catalyzes Lys63-linked ubiquitylation of 53BP1 required for the initial localization of 53BP1 at damage sites (34). Additionally, Thorslund et al (2015) reported that overexpression of RNF168 is sufficient to restore 53BP1 recruitment at DSBs in cells that can no longer synthesize Lys63-linked ubiquitin chains (6).…”

RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

“…52), support the importance of HR in the repair of etoposide-induced DSBs. Interestingly, in contrast to BRCA1, RNF168 deficiency only mildly affects HR-mediated DSB repair36. Therefore, the difference in the requirement for BRCA1 and RNF168 for HR-mediated repair could also contribute to the different response of BRCA1 and RNF168 deficient cells to TOP2 catalytic inhibitors and poisons.…”

RNF168 Promotes Noncanonical K27 Ubiquitination to Signal DNA Damage

“…One candidate to be investigated is 53BP1, because it has been recently demonstrated to be targeted by RNF168's activity (Bohgaki et al, 2013). Another interesting candidate is the polycomb protein Ring1b.…”