The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

Abstract

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.

The BRUCE‐ATR Signaling Axis Is Required for Accurate DNA Replication and Suppression of Liver Cancer Development

“…C), which was verified to be BRUCE‐specific using a reconstitution experiment involving our published cell lines (Fig. D,E) . Together, these results demonstrate that BRUCE‐dependent activation of ATR is required for signal transmission to its downstream target FANCD2.…”

“…Active ATR kinase phosphorylates CHK1 at S345, RPA32 at S33 (the small subunit of the RPA complex), and a variety of other proteins through which ATR activation counters replication stress. BRUCE levels were depleted in U2OS cells by DOX‐inducible short hairpin BRUCE (shBRUCE), as previously described . Subsequent western blot analysis indicated that BRUCE depletion attenuated ATR‐dependent phosphorylation of CHK1 at S345 (pCHK1‐S345) and RPA32 at S33 (pRPA32‐S33) following induction of replication stress by either MMC or HU treatment (Fig.…”

“…BRUCE is a large scaffold protein that interacts with other proteins to orchestrate cellular functions . To gain mechanistic insight into how BRUCE regulates ATR signaling, we took an unbiased approach to search for BRUCE‐interacting proteins.…”

“…Importantly, we have discovered that BRUCE promotes ATM activation and permits efficient access of multiple DDR factors to repair DNA DSBs through homologous recombination repair. Additionally, we found that BRUCE‐deficient cells display spontaneous chromosomal breaks and gaps …”

“…Genome instability can be induced by DNA DSBs and replication stress, both of which can generate the spontaneous chromosomal abnormalities we observed in BRUCE‐deficient cells . In this study, we used biochemistry, cell biology, and proteomic approaches to assess the function of BRUCE in ATR signaling under replication stress conditions.…”