Date of Award

Summer 2021

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Jason Stewart

Abstract

Genomic DNA is constantly damaged by both internal and external genotoxins and cellular processes. A consequence of unrepaired or misrepaired DNA damage is genome instability, a hallmark of cancer and numerous other diseases. Eukaryotic cells have developed complex mechanisms to repair DNA damage, which is collectively referred to as a DNA damage response (DDR). CST (CTC1-STN1-TEN1) is a heterotrimeric, RPA-like protein complex that binds to single stranded (ss)DNA. In humans, CST functions in the replication and maintenance of telomeres, structures found at the ends of linear chromosomes, as well as non-telomeric DNA. Previous studies showed that deletion of the largest CST subunit, CTC1, results in decreased cell proliferation and telomeric DNA damage signaling. However, the consequences of conditional CTC1 or STN1 knockout (KO) at the cellular level have not been fully elucidated. Consistent with previous findings, we demonstrate that CTC1 or STN1 KO results in decreased cell proliferation, G2 arrest and RPA-bound telomeric ssDNA, which should trigger a DDR through the ATR-CHK1 pathway. However, despite the increased levels of telomeric RPA-ssDNA, global ATR-dependent CHK1 and p53 phosphorylation was not detected in CTC1 and STN1 KO cells. Interestingly, we show that RPA-ssDNA still activates ATR, leading to the phosphorylation of RPA and autophosphorylation of ATR, and that G2 arrest is dependent on exhaustion of the telomere protection factor, POT1 in CTC1 KO cells. These results suggest that ATR is localized and active at RPA-bound telomeres but is unable to elicit a global checkpoint response through CHK1 viii Furthermore, CTC1 or STN1 KO inhibited CHK1 phosphorylation following replication stress due to decreased levels of the ATR activator TopBP1. Finally, we investigated whether the phenotypes caused by CTC1 deletion can potentially be rescued by various CTC1 mutant constructs. Preliminary studies find that TopBP1 levels can be restored with different mutant versions of CTC1, providing clues to how different CST domains may regulate the DDR. Overall, our results identify CST as a novel regulator of the ATR-CHK1 pathway.

Rights

© 2021, Stephanie Michelle Ackerson

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