The long-term goal of this project is to define the molecular mechanisms underlying the eukaryotic DNA damage and DNA replication checkpoint responses. These pathways are critical in coordinating cell cycle progression with DNA metabolism, and defects in their function can lead to genetic instability and resultant cell death or transformation. The importance of this coordination in preventing carcinogenesis has been illustrated by studies on p53, a protein that is mutated in a majority of cancers and that plays a key role in the DNA damage checkpoint response. Genetic evidence indicates that the eukaryotic checkpoint pathways have been highly conserved through evolution, allowing for the use of flexible model systems such as yeast to gain information that will be useful for cancer treatment. One conserved checkpoint pathway includes ATM, the protein mutated in ataxia-telangiectasia (A-T), and replication protein A (RPA), the cellular single-stranded DNA-binding protein. A-T cells are defective in the DNA damage checkpoint response and in ionizing radiation-induced RPA phosphorylation. Similar checkpoint- and phosphorylation-defective phenotypes result from mutation of Mec1p, a budding yeast equivalent of ATM. The objective of this application is to characterize the mechanism and function of Mec1p-dependent RPA phosphorylation. The central hypothesis underlying the proposed research is that Mec1p-dependent RPA phosphorylation is a recombination regulatory mechanism.
The specific aims of the proposed study are to 1) characterize Mec1p- dependent RPA kinase activity, 2) characterize phosphorylated RPA, and 3) define functions of the Mec1p/RPA pathway. In order to achieve these goals, biochemical methods that have been used to characterize fundamental eukaryotic processes such as DNA replication, repair, and recombination will be employed. Mec1p- dependent protein kinase activity will be purified from yeast cells and characterized physically and enzymatically. In addition, phosphorylated RPA will be purified and compared with unphosphorylated RPA for both structure and nucleic acid-binding activity. Finally, both genetic and biochemical techniques will be employed to determine the physiological function of Mec1p- dependent RPA phosphorylation. The principal investigator is uniquely qualified to pursue these goals because he has extensive experience in genetic and biochemical experimentation and in the study of Mec1p and RPA. It is expected that these studies will provide significant insight into the molecular mechanisms underlying maintenance of the non-cancerous state under genotoxic conditions.
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