| Summary: | Telomeres are defined as the repetitive sequences at the end of linear chromosomes. The loss of telomere integrity leads to genome instability. Telomere integrity is maintained by the coordination of replication, heterochromatin, and DNA damage repair machinery. In this thesis, I describe how Origin Recognition Complex-associated (ORCA/LRWD1), regulates telomere integrity in Alternative Lengthening of Telomeres (ALT) -positive cells. In the absence of Telomere Maintenance Mechanism (TMM), the end replication problem will lead to the loss of telomere length through each round of cell division, and eventually to senescence. In order to maintain telomere length, stem cells and most cancer cells activate telomerase. The remaining subset of cancer cells utilizes a homologous recombination (HR) -based mechanism, the Alternative Lengthening of Telomeres (ALT). The current understanding for ALT-telomeres include: 1) break-induced replication is essential for ALT-telomere maintenance; 2) ALT-telomeres contain less condensed chromatin; and 3) ALT-telomeres are prone to replication stress. The detailed mechanism for ALT remains elusive. Previous studies from our lab have shown that ORCA is essential for DNA replication initiation and heterochromatin organization. Interestingly, ORCA, along with other subunits of ORC, is enriched at ALT-telomeres. Why ORCA is enriched at ALT-telomeres, and the functional relevance of this enrichment, remain unknown. In Chapter 2, I describe that LRWD1/ORCA regulates ALT-activity through modulating heterochromatin and RPA binding to ssDNA. I identified that the SUMOylation of shelterin components in essential for ORCA recruitment to ALT-telomeres. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of Homologous Recombination (HR), RPA and RAD51, and consistent with this, we observe increased ALT-associated Promyelocytic leukemia body (APB) formation, c-circle level, and telomere sister chromatid exchange (T-SCE). The loss of ORCA induced overall increase in HR, suggesting that ORCA functions as a negative regulator for HR. The function of ORCA at ALT-telomeres is ORC-independent. ORCA directly binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction. In Chapter 3, I summarize my findings on how ORCA regulates ALT-activity. I also discuss the interesting findings in my research, and how these aspects could lead to further explorations of the roles of ORCA in centromere integrity and DNA damage response. U of I Only Author requested U of Illinois access only (OA after 2yrs) in Vireo ETD system
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