Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

8-2013

Committee Chair or Co-Chairs

Yue Zou

Committee Members

Phillip R. Musich, Antonio E. Rusinol, Michelle M. Duffourc, William L. Stone

Abstract

Human replication protein A (RPA) is an eukaryotic single-stranded DNA binding protein directly involved in a variety of DNA metabolic pathways including replication, recombination, DNA damage checkpoints and signaling, as well as all DNA repair pathways. This project presents 2 novel roles of RPA in the cellular response to DNA damage. The first elucidates the regulation of RPA and p53 interaction by DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) in homologous recombination (HR). HR and nonhomologous end joining (NHEJ) are 2 distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-PK, the core component of NHEJ, partners with DNA-damage checkpoint kinases ATM, and ATR to synergistically regulate HR repair of DSBs. The regulation was accomplished through modulation of the p53-RPA interaction. We show that upon DNA damage p53 and RPA are freed from the p53–RPA complex. This is done through simultaneous phosphorylation of RPA by DNA-PK, and p53 by ATR and ATM. Neither the phosphorylation of RPA nor that of p53 alone could dissociate the p53-RPA complex; furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR and NHEJ repair through the coregulation of p53–RPA interaction by DNA-PK, ATM and ATR.

The second part of this project reveals a novel role of RPA32 phosphorylation in suppressing the signaling of programmed cell death, also known as apoptosis. Our results show that deficiency in RPA32 phosphorylation leads to increased apoptosis after genotoxic stress. Specifically, PARP-1 cleavage, Caspase-3 activation, sub-G1 cell population, annexin V staining and the loss of mitochondrial membrane potential were significantly increased in the phospho-deficient RPA32 cells (PD-RPA32). The lack of RPA phosphorylation also promoted activation of initiator Caspase-9 and effector Caspase-3 and -7. This regulation is dependent on the kinase activity of DNA-PK and is mediated by PUMA through the ATM-p53 pathway. Our results suggest a novel role of RPA phosphorylation in apoptosis that illuminates a new target that lies on the crossroads of DNA repair and cell death, a pivotal point that could be of importance for sensitizing cancer cells to chemotherapy.

Document Type

Dissertation - unrestricted

Copyright

Copyright by the authors.

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