BRIT1/MCPH1 MEDIATES THE DNA DAMAGE RESPONSE BY INDUCING P53 STABILITY AND PROMOTING ATR SIGNALING
Date of Graduation
Doctor of Philosophy (PhD)
The BRCT-repeat inhibitor of hTERT (BRIT1)/MCPH1 protein promotes the process of homologous recombination (HR) to repair DNA double strand breaks (DSBs). In response to DSBs, BRIT1 foci form at damaged sites, and recruits downstream repair proteins including 53BP1, MDC1, NBS1, and the SWI/SNF complex to the DSB region to promote DNA repair. BRIT1 copy number deficiency correlates with increased genomic instability in ovarian cancer specimens and breast cancer cell lines. Here, we propose that additional functions of BRIT1 include a direct interaction with the p53 tumor suppressor protein to promote p53 stability, and binding and recruitment of TopBP1 to sites of replication stress to maintain ATR signaling.
The stability of p53 is largely dependent on its negative regulator, the MDM2 ubiquitin E3 ligase. Here, we provide evidence that in addition to its role as an upstream regulator of DNA damage response, BRIT1 is able to directly interact with p53 to promote p53 stability. Our results suggest that BRIT1 protects p53 from degradation, and this occurs even in the absence of DNA damage. The level of BRIT1 expression proportionally reduces MDM2-p53 interaction, increases MDM2 ubiquitylation and degradation, while decrease the ubiquitylation of p53. We show that these findings are clinically relevant, as BRIT1 deficiency increases breast cancer cell proliferation and transformation in vitro, while BRIT1 expression reduces breast tumor growth in vivo.
The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a major mediator of DNA damage repair and replication stress response pathways. The key function of ATR is to induce S and G2 phase checkpoints through its interaction and phosphorylation of several proteins, including Chk1. We demonstrate here that BRIT1 is required to maintain ATR activation for cellular recovery from DNA damage and replication stress. We found that BRIT1 directly interacts with TopBP1. BRIT1 deficiency compromises TopBP1 foci formation, and reduces activation of ATR targets. We also found that the BRIT1-TopBP1 interaction is dependent on ATM/ATR-mediated phosphorylation of BRIT1 at Ser322, suggesting that ATM/ATR instigate a feed-forward mechanism through BRIT1, allowing long-term activation of DNA repair. Together, our work provides insight into novel mechanism for how BRIT1 promotes genome stability.
BRIT1, MCPH1, ATR signaling, replication stress response, TopBP1, ATM, p53, MDM2, breast cancer, DNA damage