Dynamics of DNA Recognition by DNA Repair Polymerase

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DESCRIPTION (provided by applicant): DNA replication and repair are fundamental processes for transmission of genetic information from one cell generation to the other and for defending the cell against damages in its DNA. At the heart of these processes is the synthesis of DNA catalyzed by the DNA polymerases. Polymerase 13 provides an outstanding model system to study the molecular mechanism of the polymerase action, due to its simplified catalytic repertoire and role in human DNA repair. The enzyme is involved in gap filling synthesis, mismatch repair, the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA. Mutations and deletions in pol 13 have been implicated in several human cancers and genetic diseases including breast, prostate, kidney, lung, colorectal cancers, and Wemer's syndrome. In light of its key role in human DNA repair, it is of fundamental importance to understand the molecular mechanism by which pol 13 functions in performing its activities. Knowledge of the mechanistic details of the enzyme mechanism is essential to our understanding of the DNA repair process in a human cell and the mechanism by which the cell defends itself against diseases. Studying different steps at the molecular level will provide the knowledge about how to control them. In turn, this knowledge will be of paramount importance in designing efficient therapies for diseases. Polymerase 13 lacks error-correcting activities typical for replicative polymerases. This indicates that DNA and dNTP recognition, which controls the fidelity of DNA syntheses, must precede the catalysis. Moreover, a profound difference between the replicative and repair polymerase is that the DNA repair enzyme must recognize a specific structure of the damaged DNA prior to the catalysis, in the context of overwhelmingly dsDNA conformation. Thus, elucidations of the energetics and dynamics of pol 13-DNA recognition processes and the structures of the formed complexes are prerequisites for understanding the molecular mechanism of the enzyme, particularly, the efficiency and fidelity of the DNA synthesis. The main goal of this project is to establish a molecular model of the recognition of specific DNA structures by pol 13. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of its complexes with DNA substrates and dNTPs in solution, using fluorescence titrations, analytical centrifugation, fluorescence stopped-flow, temperature-jump, rapid-quench-flow, fluorescence energy transfer, and site-directed mutagenesis techniques.