Molecular Recognition At Dna Damage Sites
BRCA1 is frequently mutated in breast and ovarian cancer patients and it exerts its tumor suppressive function within several distinctive complexes by facilitating error-free DNA repair via homologous recombination (HR) mechanism. The particular focus of this dissertation is the BRCA1-RAP80 ubiquitin recognition complex, which is composed of five core constituents (RAP80, Abraxas, MERIT40, BRCC45, and BRCC36) and targets BRCA1 to the chromatin flanking DNA damage sites. Although this complex is required for BRCA1 chromatin localization, its physiologic role has remained enigmatic, as has its relationship to canonical DNA repair mechanisms. Here we show that Merit40 (scaffolding protein of the RAP80 complex) deficient mice displayed marked hypersensitivity to DNA inter-strand crosslinks (ICLs), but not to whole body irradiation. Instead, Merit40 mutation exacerbated ICL induced chromosome instability in the context of concomitant Brca2 deficiency. These findings define specific functional interactions between the RAP80 complex dependent ubiquitin recognition and the FA-BRCA ICL repair network. As destabilization of the RAP80 complex is well tolerated in mice and could sensitive Brca2 mutant cells to frequently used chemotherapeutic agent mitomycin C (ICL-inducing agent), manipulating this complex might allow selective killing of Brca2 cancer cells. Moreover, inhibiting the enzymatic activity of the complex member BRCC36 was able to recapitulate Merit40 deficiency in cells and cause increased lethality in Brca2 mutant mice, indicating the RAP80 complex mediates ICL repair via the deubiquitinating (DUB) enzymatic activity of BRCC36. Mechanistically, we revealed that RAP80 is a substrate of BRCC36 and this deubiquitination process is essential to regulate the BRCA1-RAP80 chromatin localization. Additionally, to understand the specific chromatin environment that recruits the RAP80 complex to DNA damage sites, we devised a novel methodology to purify mono-nucleosomes bound by chromatin associated DNA repair proteins and employed mass spectrometry to quantitatively measure the abundance of individual post-translational modifications on these nucleosomes. This study allows us to assess the full spectrum of chromatin modifications that associate with different DNA repair pathways, and thus to direct DNA repair pathways by manipulating enzymes that are responsible for these modifications.
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