Doctor of Philosophy, Case Western Reserve University, 2024, Pharmacology

Characterized as chromatin with repressive transcription, heterochromatin has been shown to protect DNA from damage and maintain genome integrity. However, due to its compact nature, how heterochromatin is maintained and plays its functions remains poorly understood. Recent studies suggest that several heterochromatin factors, such as HP1α, KAP1, and SUV39H1/2, could drive the soluble heterochromatin into a phase-separated droplet, called liquid-liquid phase separation (LLPS). This property limits the components necessary for heterochromatin function accessing the droplets. In this study, we found P53 binding protein 1 (53BP1), a multiple-domain protein facilitating DNA double-strand break (DSB) repair, to be one of the heterochromatin factors with LLPS properties. 53BP1 is critical in maintaining heterochromatin via LLPS, independent of its DSB repair function. In addition, we found that 53BP1 interacted with factors involved in DNA replication and epigenetic modification in heterochromatin condensates through a special crosslinking coupled mass spectrometry. 53BP1 deficiency impaired heterochromatin mark restoration post-replication, delaying the overall S phase progression. We then directed our attention toward one of the critical factors, proliferating cell nuclear antigen (PCNA). We carried out a detailed analysis of the interaction between PCNA and 53BP1 and figured out the role of such interaction in 53BP1 LLPS, DSB repair, and heterochromatin replication. These findings reinforce that 53BP1 governs heterochromatin replication by restoring epigenetic modifications in condensate states. Our studies provide a foundation for future investigations targeting 53BP1 LLPS to fulfill the therapeutic need for heterochromatin-related diseases.

Committee: Youwei Zhang (Advisor); Johannes Von Lintig (Committee Chair); John J. Mieyal (Committee Member); Witold Surewicz (Committee Member); Zhenghe J Wang (Committee Member); Zihua Gong (Committee Member) Subjects: Biomedical Research; Cellular Biology

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