Biochemical and functional characterization of the tumor suppressors BRCA1 and BAP1
Ubiquitination is a post-translational modification that plays major roles in regulating a plethora of cellular processes. In this thesis, I will discuss the biochemical and functional characterization of two functionally related proteins, BRCA1 and BAP1, both of which are important tumor suppressors. BRCA1, an ubiquitin ligase that catalyzes the attachment of ubiquitin to target proteins, is mutated in breast and ovarian cancers. BRCA1 roles in maintaining genomic stability following DNA double strand breaks (DSBs) by promoting the homologous recombination repair pathway is well established. However, how BRCA1 is regulated following genotoxic stress that does not directly involve DSBs is still not fully elucidated. We showed that BRCA1 is downregulated, through proteasomal degradation, following exposure of the cells to the DNA helix distorting agent UV or the DNA alkylating agent Methyl Methanesulfonate (MMS), two DNA damaging agents that do not directly generate DSBs. BRCA1 downregulation is reversible and is independent of the PI3 kinase related kinases, ATM, ATR or DNA-PK which constitute primary responders that are rapidly activated by DNA damage. We proposed that BRCA1 downregulation prevents the untimely recruitment of BRCA1 and associated factors to DNA damage sites that are not DSBs, thus coordinating the DNA damage/repair response.   The deubiquitinating enzyme BAP1 was initially identified as an interacting protein that regulates the function of BRCA1. BAP1 is also known to interact with the Polycomb group proteins ASXL1 and ASXL2. However, the importance of this interaction was not fully understood. We showed that BAP1 forms two mutually exclusive complexes with ASXL1 and ASXL2. These interactions are critical for BAP1 binding to ubiquitin and stimulation of its deubiquitinase activity towards histone H2A. We also identified cancer-derived mutations of BAP1 that abrogate its interaction with ASXL1 and ASXL2 and deubiquitinase activity, which provide a direct mechanistic link between H2A deubiquitination and tumorigenesis.   Elucidating how BRCA1 and BAP1 are regulated will lead to a better understanding of their roles as tumor suppressors and this will in turn help establishing improved diagnostic and therapeutic strategies to treat cancer.
Ubiquitination BRCA1 deubiquitination BAP1 ASXL1 ASXL2 histone H2A DNA damage