The incorporation of therapeutic nucleoside analogs (ara-C, gemcitabine, fludarabine) into DNA is the critical event in causing cytotoxicity to cancer cells. Excision of the incorporated analogs from DNA represents a key mechanism by which cells resist the drug action. However, the cellular molecule(s) responsible for the removal of incorporated drugs from DNA has not been identified. The 3'-5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule but its biological significance remains elusive. The applicant's preliminary studies demonstrated that the 3'-5' exonuclease activity of the wild-type p53 protein preferentially removes mismatched nucleotides from the replicating DNA strand and significantly enhances DNA replication fidelity. Furthermore, in vitro studies indicate that the wild-type p53, but not mutant p53, effectively excises the incorporated nucleoside analog gemcitabine monophosphate from DNA. The objectives of this research project are to systematically characterize the 3'-5' exonuclease of p53 in the excision of mismatched nucleotides and incorporated therapeutic analogs from DNA, and to investigate the biological significance of this exonucleolytic activity. The applicant will test the hypothesis that wild-type p53 enhances DNA replication fidelity by preferential removal of mismatched nucleotides from DNA and confers cellular resistance to therapeutic analogs by direct excision of the incorporated drugs from DNA. Biochemical approaches will be combined with molecular strategies to test this hypothesis. The ability of the wild-type p53 and different mutant p53 proteins to excise mismatched deoxynucleotides and nucleotide analogs from DNA will first be tested and quantitatively evaluated in vitro using a variety of defined DNA templates. The biological relevance of this exonucleolytic function will be further investigated in whole cells with different genetic makeup of p53. This research project will provide new insights into the biochemical mechanisms by which wild type p53 guards the integrity of DNA and modulates the sensitivity of cells to anticancer nucleoside analogs. Furthermore, the inability of mutant p53 to remove nucleoside analogs from DNA may provide a molecular basis for the design of strategies to selectively kill cancer cells with mutant p53 by therapeutic nucleoside analogs.
Showing the most recent 10 out of 15 publications
