DOI
https://doi.org/10.25772/6VDG-6383
Defense Date
2014
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
Kristoffer Valerie
Abstract
When cultured in vitro, human embryonic stem cells (hESCs) acquire genetic abnormalities that have slowed their therapeutic use. As hESCs have a “leaky” G1/S boundary, the pressure of ensuring genetic integrity falls on the G2/M checkpoint, which can be activated by failed chromosomal decatenation (among other stimuli). It is hypothesized that hESCs have a deficient decatenation checkpoint, but little data supports this. Evidence suggests that the ataxia telangiectasia mutated (ATM) kinase controls the G2/M decatenation and DNA damage checkpoints, though previous reports are conflicting on this point. My work demonstrates that inhibition of decatenation activates ATM and arrests hESCs in G2. Pharmacologic inhibition of ATM (ATMi) abrogates this arrest, allowing hESCs to enter mitosis. Live cell imaging studies reveal that ATMi increases the time it takes to complete mitosis. Culture of cells under ATMi causes a gain of DNA content, which is reversed once ATMi is relieved. BRCA1, a known target of ATM, is also involved in the G2/M checkpoint. Experimental evidence reveals that activated ATM phosphorylates BRCA1, preventing Aurora A from interacting with and phosphorylating BRCA1 on S308, a modification necessary for mitotic entry. Together, this data illuminates a novel pathway by which ATM activation mediates G2 arrest in hESCs.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
11-18-2013