Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

In this dissertation study, we have investigated the protein functions in DNA double-strand break (DSB) repair of three important factors, BRCA1, 53BP1 and SUMO isoforms, at levels of biochemical activity, protein dynamics and chromosomal DNA repair. Our work reveals novel mechanisms of these proteins functioning in response to DSB damage, hence providing insights of where and how they are actively involved in each subpathway of DSB repair. In the first part of our work, we studied BRCA1, a tumor suppressor important for the maintenance of genomic stability including centrosome control and DSB repair, and found that a putative enzymatic mutant of BRCA1— BRCA1(I26A), which had been thought to disrupt its E3 ligase activity, was still functional in the cellular processes of regulating centrosome number and homologous recombination-dependent DSB repair, thereby raising a question of whether I26A mutant is indeed inert. Reevaluation of the ubiquitination activity of this BRCA1(I26A) mutant revealed that it is an active E3 ubiquitin ligase when associated with the appropriate E2 factor. We then think that conclusions about the dispensability of the BRCA1-dependent enzymatic activity in various cellular processes should be reconsidered. Next we studied the unique function of 53BP1, a known NHEJ factor for DSB repair. We found that 53BP1 specifically promotes the error-free conservative-NHEJ (C-NHEJ) mechanism, dependent on its upstream recruiters RNF8 and RNF168. 53BP1 has no effect on the highly mutagenic and deletional alternative-NHEJ (Alt-NHEJ) pathway or on homology-directed repair (HDR), but it suppresses single-strand annealing (SSA). We discovered that the localization of 53BP1 at sites of DSBs is accompanied by its bulk removal from the nucleus except at sites of DNA damage. And the degradation of bulk 53BP1 upon DNA damage is due to each action of RNF8 and RNF168. Further, we showed that failure to degrade bulk 53BP1 results in the failure for its downstream (open full item for complete abstract)

Committee: Jeffrey Parvin (Advisor); Altaf Wani (Committee Member); Qianben Wang (Committee Member); Robin wharton (Committee Member) Subjects: Molecular Biology