Every cell divisions cycle, over 6.8 billion base pairs of DNA must be accurately replicated. To further complicate this process, the DNA is damaged at a rate of ~10,000 lesions per cell per day. To overcome these obstacles, the kinase ATR (Ataxia telangiectasia mutated and RAD3 related) activates every S-phase. ATR maintains genomic integrity through regulation of origin firing, promotion of DNA repair, stabilization of stalled replication forks, modulation of the cell cycle, and regulation of senescence and apoptosis. The activation of ATR is a multistep process and results in phosphorylation of hundreds of downstream substrates. The consequences of which remain mostly undefined. Additionally, many common DNA-damaging chemotherapies activate ATR. As such, ATR is a promising new cancer therapeutic target. Recently developed ATR inhibitors are currently entering phase II clinical trials, however which patients should be treated with these compounds remains unknown.
In my dissertation work, I completed several projects, which revolved around modulation of ATR kinase activity. I examined the consequences of ATR inhibition on the replication fork on a single molecule level. ATR inhibition results in rapid slowing of the replication fork and increased origin firing, and in the presence of replication stress and ATR inhibition, replication forks collapse. Further work examining potential ATR autophosphorylation sites identified point mutations dramatically altering the kinase activity. These point mutations suggest the conformational changes undergone within ATR during activation of the kinase involve the HEAT repeats. Last, a whole genome siRNA screen was conducted in combination with the ATR inhibitor to identify new ATR pathway genes as well as identify clinically actionable synthetic lethal relationships. We identified the ATR pathway and DNA replication as the top pathways when lost that result in increased sensitivity to the ATR inhibitor. Our work indicates patients with cancers containing ATR pathway defects will have increased sensitivity to the ATR inhibitor alone or in combination with cisplatin.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07152016-092619 |
| Date | 15 July 2016 |
| Creators | Luzwick, Jessica W |
| Contributors | Scott Hiebert, David Cortez, Stephen Fesik, William Tansey, Jennifer Pietenpol |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07152016-092619/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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