Ubiquitylation in DNA double-strand break repair

Abstract

Genome integrity is constantly challenged by various DNA lesions with DNA double-strand breaks (DSBs) as the most cytotoxic lesions. In order to faithfully repair DSBs, DNA damage response (DDR) signaling networks have evolved, which organize many multi-protein complexes to deal with the encountered DNA damage. Spatiotemporal dynamics of these protein complexes at DSBs are mainly modulated by post-translational modifications (PTMs). One of the most well-studied PTMs in DDR is ubiquitylation which can orchestrate cellular responses to DSBs, promote accurate DNA repair, and maintain genome integrity. Here, we summarize the recent advances of ubiquitin-dependent signaling in DDR and discuss how ubiquitylation crosstalks with other PTMs to control fundamental biological processes in DSB repair.

Keywords: DNA damage response; Double strand break; homologous recombination (HR); non-homologous end joining (NHEJ); ubiquitylation.

Fig.1. Diversity of mechanisms involving ubiquitin conjugation.

Ub can be covalently conjugated to specific substrate in a three-step enzymatic cascade involving ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3). The substrate can be modified with a single ubiquitin molecule (monoubiquitylation) or multi-ubiquitin formed polyubiquitin chain (polyubiquitylation). The amino-terminal methionine (M1) and lysines at K6, K11, K27, K29, K33, K48 and K63 can serve as acceptor sites for the formation of specific polyubiquitin chain. Deubiquitylating enzymes (DUBs) remove ubiquitin from substrates, making ubiquitylation a highly dynamic and reversible process.

Fig.2. Ubiquitin-dependent protein assembly at DSBs and their roles in DSB repair pathway choice.

Ubiquitin-dependent signaling cascade plays important roles in recruiting DDR fators to sites of DSB. This recruitment relies on K63-linked ubiquitylation of histones and other chromatin-binding proteins through RNF8- and RNF168-dependent pathway. DSBs are first recognized by MRN complex, triggering the recruitment and activation of ATM. ATM phosphorylates H2AX at Ser139 (known as γH2AX), generating a binding site for MDC1. ATM-dependent phosphorylation of L3MBTL2 promotes its interaction with MDC1 and its recruitment to sites of DSB. Subsequently L3MBTL2 is ubiquitylated by RNF8 which recruits RNF168 to sites of DNA damage. RNF168, in turn, monoubiquitylates H2A to amplify the DNA damage signal and promote the engagement of ubiquitin-binding domain (UBD)-containing repair factors for proper DSB signaling and DSB repair. DNA end resection plays a crucial role in dictating DSB repair pathway choice. By recognizing both monoubiquitylated H2A K15 and dimethylated H4K20, 53BP1 is recruited to DSBs to antagonize BRCA1-dependent end resection via ATM phosphorylation-dependent interactions of 53BP1 with its binding partners RIF1 and PTIP. PTIP recruits Artermis which trims DNA end and promote NHEJ. RIF1 recruits REV7, the Shielding complex (SHLD1–SHLD2–SHLD3) and the CST/Pol α-Prim complex to shield DNA end from BRCA1 dependent end resetion, thus promote NHEJ. Conversely, BRCA1 antagonizes NHEJ by impairing RIF1 retention at DSBs in S phase in a CtIP-dependent manner. BRCA1 is recruited by RAP80 which is initially recruited to DSBs through the binding to K63- or K6-linked polyubiquitin chains. HR is initiated with two-step DNA end resection by MRN-CtIP and DNA2-EXO1 in 5’−3’ direction to generate a long 3’ ssDNA overhang which is rapidly coated by replication protein A (RPA). With the help of mediator complex BRCA1-PALB2-BRCA2, RAD51 displaces RPA to form RAD51-ssDNA filaments which perform homology search and strand invasion followed by DNA synthesis at the resected strand. RNF138 is required for stimulating CtIP ubiquitylation and accrual at DSB sites. Multi-site ubiquitylation of RPA by ubiquitin ligases RFWD3 and PRP19 facilitates homologous recombination by as-yet-unknown mechanisms. CRL3^KEAP1 can ubiquitylate PALB2 to suppress its interaction with BRCA1 and HR and this ubiquitylation is counteracted by USP11. The E3 ligase UHRF1 can mediate K63-linked polyubiquitination of RIF1 which leads to its dissociation from 53BP1 and DSBs, thereby promoting HR.

Fig.3. Simplified illustration of the crosstalk of ubiquitylation with other PTMs following DSB induction.

See text for details.