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

Available for download on Thursday, July 11, 2019

Included in

Biochemistry Commons

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