Mammalian DNA polymerase beta catalyzes abasic site excision and nucleotidyl transfer in DNA repair and replication in mammalian cells. The enzyme is central to the repair of environmentally induced DNA damage in humans. Characterization of the structural mechanism and the factors leading to DNA replication fidelity by beta-Pol are highly important for understanding the function and/or dysfunction of this enzyme. Misincorporations by beta-Pol within single-stranded DNA prone to replication errors relate directly to the structural mechanism of template-primer binding or DNA adduct binding by the enzyme and potentially lead to mutations within genomic DNA and disease. The solution structure, DNA interactions, and backbone dynamics of the N- terminal (residues 2-87) and C-terminal (residues 88-334) domains of beta-Pol are being characterized by nuclear magnetic resonance (NMR) methods. The N-terminal and C-terminal domains are readily obtained with 15N/13C labeling in yields of 40-50 mg of protein domain from 2 liters of culture medium. Studies completed for the N-terminal domain have included the determination of a highly refined solution structure, characterization of the template ssDNA interaction interface, and determination of the backbone dynamics. Backbone dynamics of DNA complexes will be studied. The products of abasic site excision by the N-terminal domain will be determined. The Schiff's base lysine, that contributes to abasic site excision activity, and the pKas of catalytic side chains will be determined by NMR. The results of these experiments will be compared to the findings for the K35A, K68A, and K72A mutants. Binding and structural studies will be directed toward the N-terminal domain in complex with a 9-mer duplex DNA containing an abasic site analogue. Multidimensional NMR of the 15N/13C and the 15N/13C/2H labeled C-terminal domain are proposed for determination of the solution secondary structure, backbone dynamics, and conformational flexibility of the polymerase. The proposed studies are aimed at understanding at a solution structural level the multiple activities in DNA polymerase beta function and replication fidelity.
Showing the most recent 10 out of 11 publications
