Defense Date

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Kristoffer Valerie

Abstract

Central to the progression of all organisms is the maintenance of a stable genome despite continuous insults arising from genotoxic and environmental stresses. Embryonic stem cells show promise for treatment of a variety of diseases as well as for providing normal human tissue to conduct scientific research. A major obstacle for their application is that genomic instability arises in stem cells after prolonged cell culture. The most detrimental form of DNA damage is the DNA double-strand break (DSB), which is managed by cells through complex mechanisms, designated the DNA damage response. There are two major types of DSB repair; homologous recombination repair (HRR) and non-homologous end joining (NHEJ), both of which are regulated by members of the phosphatidyl-inositol-3’-kinase-related kinase (PIKK) family, including Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia Mutated and Rad3-related (ATR) and the DNA dependent protein kinase (DNA-PK). The aim of this study was to define the mechanisms and important proteins involved in repair of human embryonic stem cells. Here we have also described a system to differentiate hESCs into neural progenitors and astrocytes and were able to examine their DNA damage response. In both examining DNA repair markers and using a DNA repair reporter assay, this work shows that ATR is involved in DSB repair early in development, whereas ATM is essential in DSB repair in differentiated cells. We also show that HRR, a high fidelity form of repair, is used extensively by embryonic stem cells and HRR diminishes as cells differentiate. We also further defined the extent of NHEJ and the role of high fidelity NHEJ from the embryonic to differentiated state. These findings further the basic knowledge of repair fidelity in embryonic and mature human tissue. The data gives insight into what proteins maintain stem cell genomic stability and may be important to develop safe technologies for tissue engineering. Specifically, we have defined what DNA damage signaling pathways are used as embryologic cells progress to a mature, functional state.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

July 2010

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