Abstract

We propose to investigate the functional significance of DNA-damage-induced, ATM/ATR-dependent phosphoryIation of the major cellular single-stranded DNA-binding protein, RPA. Since RPA is essential for DNA replication and DNA repair, phosphorylation-dependent alteration in RPA function has the potential of altering the cellular responses to DNA damage. Indeed, we have demonstrated that DNA damage-induced ATM-dependent phosphorylation of RPA occurs, and it changes RPA function in vitro. Specifically, RPA phosphorylation alters DNA binding activity and protein/protein interactions. We postulate that a failure to phosphorylate RPA and other proteins required for DNA replication and DNA repair in ATM mutants compromises the cellular switch from DNA replication to DNA repair that would normally protect cells from the deleterious consequences of DNA damage. We propose that these abnormal responses in A-T lead to the enhanced cell death and genetic instability associated with the disease. We propose to test the hypothesis that the failure to phosphorvlate RPA (and other DNA repair proteins) in response to DNA damage compromises the DNA repair capacity of A-T ceils.
The specific aims of this project are: 1. To determine the sites of ATM- and ATR-mediated RPA phosphorylation and construct site-specific mutants to test their role in RPA function. 2. To determine the influence of RPA (and mutant RPA) phosphorylation on protein/protein interactions in vivo and in vitro. A newly-developed RPA phospho-specific antibody will greatly facilitate these studies. 3. To clarify the role of the ATM kinase activity vs. other ATM functions in DSB repair: We are concentrating on abnormal responses to DNA damage in A-T cells that may influence the DNA repair capacity of these cells and may contribute to the high cancer risk and neurodegeneration. The ultimate goal of this project is to improve our understanding of the mechanisms that cells use to maintain genomic stability. This improved understanding may ultimately lead to the development of strategies for preventing and/or treating diseases that are characterized by genomic instability and neurological defects.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
7R01NS034782-08
Application #
7011365
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Tagle, Danilo A
Project Start
1997-01-01
Project End
2008-04-30
Budget Start
2004-08-01
Budget End
2005-04-30
Support Year
8
Fiscal Year
2004
Total Cost
$349,587
Indirect Cost

  Institution

Name
University of Arizona
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
806345617
City
Tucson
State
AZ
Country
United States
Zip Code
85721

  Publications

Thompson, Eric G; Fares, Hanna; Dixon, Kathleen (2012) BRCA1 requirement for the fidelity of plasmid DNA double-strand break repair in cultured breast epithelial cells. Environ Mol Mutagen 53:32-43
Rahal, Elias A; Henricksen, Leigh A; Li, Yuling et al. (2010) ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining. Cell Cycle 9:2866-77
Rahal, Elias A; Henricksen, Leigh A; Li, Yuling et al. (2008) ATM mediates repression of DNA end-degradation in an ATP-dependent manner. DNA Repair (Amst) 7:464-75
Robison, Jacob G; Bissler, John J; Dixon, Kathleen (2007) Replication protein A is required for etoposide-induced assembly of MRE11/RAD50/NBS1 complex repair foci. Cell Cycle 6:2408-16
Robison, Jacob G; Dixon, Kathleen; Bissler, John J (2007) Cell cycle-and proteasome-dependent formation of etoposide-induced replication protein A (RPA) or Mre11/Rad50/Nbs1 (MRN) complex repair foci. Cell Cycle 6:2399-407
Patrick, Steve M; Oakley, Greg G; Dixon, Kathleen et al. (2005) DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair. Biochemistry 44:8438-48
Robison, Jacob G; Lu, Lu; Dixon, Kathleen et al. (2005) DNA lesion-specific co-localization of the Mre11/Rad50/Nbs1 (MRN) complex and replication protein A (RPA) to repair foci. J Biol Chem 280:12927-34
Nuss, Jonathan E; Patrick, Steve M; Oakley, Greg G et al. (2005) DNA damage induced hyperphosphorylation of replication protein A. 1. Identification of novel sites of phosphorylation in response to DNA damage. Biochemistry 44:8428-37
Dixon, Kathleen; Kopras, Elizabeth (2004) Genetic alterations and DNA repair in human carcinogenesis. Semin Cancer Biol 14:441-8
Robison, Jacob G; Elliott, James; Dixon, Kathleen et al. (2004) Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks. J Biol Chem 279:34802-10

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