EXCEED THE SPACE PROVIDED. Eukaryotic cells are routinely exposed to agents that cause DNA damage and arrest of DNA replication. In response, an as-yet unidentified signal activates highly-conserved signal transduction pathways, termed DNA checkpoints, that halt cell-cycle progression and promote DNA repair. The focus of this proposal is on the role of Replication Protein A (RPA) in this response. RPA is a heterotrimeric ssDNA binding protein that is found in all eukaryotic cells from yeast to humans. RPA's function in DNA replication, repair, and recombination is well understood, but its role in the DNA damage response is unclear. RPA appears to have two roles; it is needed early to fully activate the response, but is also phosphorylated by checkpoint kinases such as Mecl in yeast. The proposed experiments will address two basic questions: (1) What is the molecular signal that initiates the DNA damage response; and (2) Does phosphorylation of RPA affect its function in DNA repair? Biochemical and genetic approaches in the yeast S. cerevisiae will be used to test the following hypotheses: (1) part of the damage signal consists of RPA bound to ssDNA; and (2) phosphorylation alters RPA's ssDNA binding activity or its interaction with repair and recombination proteins.
In Aim 1 the function of damage-dependent RPA phosphorylation will be determined by identifying the sites of modification, mutating them, and testing the mutant alleles for checkpoint function in yeast. Mutant and phosphorylated forms of RPA will be assayed for altered ssDNA binding activities or altered interactions with recombination proteins.
In Aim2 the signal that initiates the DNA damage response will be examined by establishing an in-vitro assay with the Mecl kinase. We will purify Mecl, characterize its enzymatic activity, and search for activators. Various DNA substrates, in the absence and presence of RPA, will be tested for activator function. The ability of mutant or phosphorylated RPA to stimulate Mecl- and Rad53- kinases will also be assayed.
In Aim 3 potential regulatory domains of Mecl will be identified by performing a structure/ function analysis. The hypothesis that Mecl regulates separate DNA repair and checkpoint pathways will be tested genetically by searching for MEC1 alleles that affect DNA repair uniquely. These studies will provide insight into the nature of the DNA damage signal and the regulation of checkpoint kinases. The results are expected to have broad implications for the mechanism of DNA repair in human cells. PERFORMANCE SITE ========================================Section End===========================================

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Portnoy, Matthew
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Rutgers University
Schools of Arts and Sciences
New Brunswick
United States
Zip Code
Bae, Kwang-Hee; Kim, Hee-Sook; Bae, Sung-Ho et al. (2003) Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro. Nucleic Acids Res 31:3006-15
Daganzo, Sally M; Erzberger, Jan P; Lam, Wendy M et al. (2003) Structure and function of the conserved core of histone deposition protein Asf1. Curr Biol 13:2148-58
Kim, Hee-Sook; Brill, Steven J (2003) MEC1-dependent phosphorylation of yeast RPA1 in vitro. DNA Repair (Amst) 2:1321-35
Fricke, W M; Kaliraman, V; Brill, S J (2001) Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase. J Biol Chem 276:8848-55
Bastin-Shanower, S A; Brill, S J (2001) Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding. J Biol Chem 276:36446-53