Fidelity of DNA Synthesis

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DESCRIPTION (provided by applicant): Pol-4 catalyzes replication of the genome and other DNA synthetic processes in human cells. However, the precise functions of Pol-4 in replication, in repair of endogenous and environmental damage, and in recombination remain to be defined. Further, we lack information on the role of somatic mutations in Pol-4 in tumorigenesis and other disease processes. Our goals are to identify the functions of Pol-4 in DNA synthesis and the possible role of increased mutation by Pol-4 in the generation of human cancer. A major approach will be to exploit mutants of Pol-4 that incorporate mutagenic nucleotide analogs and thereby serve as tools to identify DNA synthesized by Pol-4 in vivo. We have four specific aims. In Aim 1, we will generate mutants of Pol-4 that increase incorporation of a specific nucleotide analog(s). In Aim 2, we will purify wild-type and mutant Pol-4 holoenzyme complexes and characterize their catalytic properties in detail, including their fidelity and kinetics of analog incorporation. In Aim 3, we will define the roles of Pol-4 in mammalian cells by introducing mutant Pol-4's that preferentially incorporate mutagenic nucleotide analogs and measuring induced mutation in cells undergoing DNA replication, repair and recombination. The induced mutations will identify the DNA synthesized by Pol-4. In Aim 4, we will assess the role of increased mutagenesis in tumor progression by performing serial transfer experiments to determine if mammalian cells that harbor mutator Pol-4 have a competitive advantage, and if there are nucleoside analogs that diminish this advantage. PUBLIC HEALTH RELEVANCE: Our objective is to establish the roles of DNA polymerase-4 in replication of the human genome and in repair of damage caused by endogenous and environmental agents. We will determine if mutations in DNA polymerase-4 promote genetic instability and accelerate tumor progression in model systems. We aim to identify a new class of chemotherapeutic agents that will retard tumor growth.