The goal of this proposal is to understand the role of phosphorylation of the middle subunit (RPA2) of the heterotrimeric human replication protein A (RPA;the eukaryotic single-stranded DNA-binding protein). Recent data arising from my laboratory indicates that RPA2 phosphorylation provides an important regulatory control over the cellular DNA damage response. Using a combination of approaches, we will characterize the role of RPA phosphorylation in vivo and in vitro. Specifically, we will examine the molecular basis by which hypo-phosphorylated RPA selectively associates with the replication fork, by characterization of its interaction with the MCM complex. The association of RPA with MCM will be examined, both in solution, and on synthetic DNA replication fork structures. Second, using a RPA2 replacement strategy, we will examine the effect of RPA2 phosphorylation site mutation on the loading of the recombination mediator BRCA2 and homologous recombination factors to sites of DNA replication stress and DNA damage. The effects of RPA2 mutation on BRCA2 loading in vitro and on HR rates in vivo will be determined. Third, we have found that the synergistic activity of these kinases towards RPA2 under genotoxic stress conditions modulate subsequent phosphorylation events both on the same RPA molecule (the cis pathway) and on different RPA molecules (the trans pathway). By exploring the cis pathway, we will test the hypothesis that S33 phosphorylation facilitates recruitment of cyclin A-Cdk2 to the RPA substrate in vitro. To examine the trans reaction, we will examine the effect of RPA phosphorylation on ATR dynamics in vivo. The data generated will provide insights into how cell cycle position causes DNA lesions to be channeled into different DNA repair pathways.

Public Health Relevance

We will understand the functional significance of RPA phosphorylation in the cellular response to DNA damage. The results from this work could lead to new threraputics to treat human cancers

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083185-04
Application #
8265967
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
2009-05-01
Project End
2015-02-28
Budget Start
2012-03-01
Budget End
2015-02-28
Support Year
4
Fiscal Year
2012
Total Cost
$477,333
Indirect Cost
$164,694
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa et al. (2014) Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery. J Cell Biol 206:493-507
Bhatt, Purvi; d'Avout, Claire; Kane, Naomi S et al. (2012) Specific domains of nucleolin interact with Hdm2 and antagonize Hdm2-mediated p53 ubiquitination. FEBS J 279:370-83
Borowiec, James A; Schildkraut, Carl L (2011) Open sesame: activating dormant replication origins in the mouse immunoglobulin heavy chain (Igh) locus. Curr Opin Cell Biol 23:284-92
Brocardo, Mariana G; Borowiec, James A; Henderson, Beric R (2011) Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress. Int J Biochem Cell Biol 43:1354-64
Lee, Dong-Hyun; Pan, Yunfeng; Kanner, Shlomo et al. (2010) A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination. Nat Struct Mol Biol 17:365-72
Vassin, Vitaly M; Anantha, Rachel William; Sokolova, Elena et al. (2009) Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress. J Cell Sci 122:4070-80
Anantha, Rachel William; Borowiec, James A (2009) Mitotic crisis: the unmasking of a novel role for RPA. Cell Cycle 8:357-61