DYNAMICS OF DNA RECOGNITION BY DNA REPAIR POLYMERASE

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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. The mammalian polymerase beta provides an outstanding model system to study the molecular mechanism of the polymerase action due to its simplified catalytic repertoire and its role in mammalian DNA repair. Pol beta is one of the four recognized DNA-directed polymerases of the eucaryotic nucleus. The enzyme is involved in gap filling synthesis, in mismatch repair, repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA. In light of its key role in mammalian DNA repair, it is of fundamental importance to understand the molecular mechanism by which pol beta functions. Knowledge of the mechanistic details of the polymerase 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, e.g., cancer. Studying different steps on the molecular level should provide the necessary knowledge about how to control these processes. In turn, this knowledge will be very useful in designing efficient therapies for diseases. DNA repair polymerase is designed to perform DNA synthesis on gapped DNA with vanishing gap size which suggests that the mode of enzyme interactions with nucleic acids is changing, in the course of DNA synthesis. Polymerase beta lacks error correcting activities, typical for replicative polymerases, which indicates that DNA and dNTP recognition processes, which control fidelity of DNA synthesis, precede the chemical step. Thus, elucidation of the energetics and dynamics of the substrate recognition process by pol beta, including the transitions between different binding modes, is a prerequisite for understanding the molecular mechanism of the enzyme action, particularly, the fidelity of the DNA synthesis. The main goal of this project is to establish a molecular model of the recognition process of DNA and dNTP substrates by pol beta. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of its complexes with DNA and dNTPs in solution using quantitative fluorescence titrations, analytical centrifugation methods, fluorescence stopped-flow, temperature-jump, rapid-quench-flow, fluorescence energy transfer, and site-directed mutagenesis techniques.