We have previously discovered the BRCA1-associated Protein-1 (BAP1) which was cloned in a screen for proteins which bind to the RING finger domain of BRCA1 BAP1 is a nuclear-localized, ubiquitin carboxyl- terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. Mutations in the BRCA1 RING finger defined in BRCA1 kindreds abolish BAP1 binding. BAP1 and BRCA1 are temporally and spatially co-expressed during development. The human BAP1 gene maps to chromosome 3p21.3 and suffers deletions and point mutations in human tumors that it is a tumor suppressor. However, until now, it has been unclear how BAP1 functioned in the proposed BRCA1 pathways of DNA repair, transcriptional regulation and recombination. Since BRCA1-null cells have a defect in the transcription-coupled repair (TCR) of oxidative DNA damage, we examined the role of BAP1 in TCR. The H226 small cell lung cancer cell line contains wild-type BRCA1 but is null for BAP1 due to a homozygous deletion at the 3p21.3 BAP1 locus. In preliminary data we show that BAP1-null cells have a TCR defect identical to BRCA1-null cells. Transfection of wild-type BAP1 into the BAP1-null cells completely restored the TCR defect. However, transfection of an engineered point mutant of BAP1 (Cys91Ser) or two independent human tumor-derived point mutations (Ala95Asp and G1y178Val) each of which abolishes the UCH enzymatic failed to restore TCR. Remarkably, a chimeric BAP1 molecule containing the UCH enzymatic domain of the BAP1-related gene UCHL3 (which shows wild-type UCH activity on model substrates) fused to the BRCA1 interaction region of BAP1 also failed to reconstitute TCR. Thus, we have discovered that BAP1 functions in the BRCA1 transcription-coupled repair pathway. This is the first reported function for a UCH enzyme and establishes that there is a substrate specificity among this family. We will address the mechanism(s) by which BAP1 regulates the BRCA1-dependent TCR DNA repair pathway by performing the following aims. Specifically: 1. Perform a comprehensive structure-function analysis of BAP-1 and define the molecular determinants for TCR. 2. Define the UCH activities and regulation of BAP-1 in response to DNA damage. 2. Search for proteins which are stabilized in BAP-1 complemented cells. These studies will establish a new link between nuclear-localized ubiquitin hydrolysis, DNA repair processes and tumor suppressor function.
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