The broad objective of Project 3 is to study the mechanisms responsible for the fidelity of DNA synthesis and thus to address the most fundamental questions concerning mutagenesis, a root cause of cancer. Our specific approach is to investigate the fidelity of DNA polymerase beta, a key repair polymerase, and human """"""""error-prone"""""""" DNA potymerases iota, mu and eta. Little is currently known concerning the interactions that these recently discovered error-prone polymerases have with replication accessory proteins, and we intend to investigate this issue because of its importance in determining polymerase """"""""trafficking"""""""", how one polymerase is chosen for a specific task over another. The unique aspect of Project 3 is that by closely integrating its specific aims with those proposed for the structural characterization of Pol beta in Project 1 and the the theoretical computational study in Project 2, we can test quantitative predictions for how active site amino acids govern the choice between incorporating right and wrong deoxynucleotide substrates. By providing a stringent test of theoretical-computational and structural predictions, the data will play a key role in refining the theoretical models. Project 3 investigates dNTP substrate transition state analogs to provide new mechanistic information concerning the source of free energy available to enable polymerases to distinguish right from wrong. The main experimental approach involves the use of fluorescence and rapid quench presteady state kinetic techniques to measure overall fidelity as well as individual fidelity base substitution and frameshift fidelity components. Project 3 will investigate genetic instability more generally by constructing model in vitro systems to study the effects of strand displacement synthesis on the expansion of trinucleotide repeat sequences causing neurodegenerative disease, and on the expansion of mono- and dinucleotide repeat sequences yielding frameshift mutation that cause cancer. The Program Project generally, and Experiment 3 more specifically, are timely given the resurgence of interest in the role of DNA polymerases in causing cancer. The studies in Experiment 3 on transition state analogs, taken in conjunction with the structural and computational projects, should provide practical payoffs in pharmaceutical anticancer drug design, and offer a logical framework in which to design drug intervention and prevention strategies to inhibit cancer progression.
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