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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052738-05
Application #
2771019
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1994-09-01
Project End
2001-08-31
Budget Start
1998-09-01
Budget End
1999-08-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Hu, Hong-Yu; Horton, Julie K; Gryk, Michael R et al. (2004) Identification of small molecule synthetic inhibitors of DNA polymerase beta by NMR chemical shift mapping. J Biol Chem 279:39736-44
Marintchev, Assen; Gryk, Michael R; Mullen, Gregory P (2003) Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase beta. Nucleic Acids Res 31:580-8
Gryk, Michael R; Maciejewski, Mark W; Robertson, Anthony et al. (2002) 1H, 13C and 15N resonance assignments for the perdeuterated 22 kD palm-thumb domain of DNA polymerase beta. J Biomol NMR 22:197-8
Gryk, Michael R; Marintchev, Assen; Maciejewski, Mark W et al. (2002) Mapping of the interaction interface of DNA polymerase beta with XRCC1. Structure 10:1709-20
Pan, B; Maciejewski, M W; Marintchev, A et al. (2001) Solution structure of the catalytic domain of gammadelta resolvase. Implications for the mechanism of catalysis. J Mol Biol 310:1089-107
Maciejewski, M W; Pan, B; Shin, R et al. (2001) 1H, 15N, and 13C resonance assignments for a 20 kDa DNA polymerase from African swine fever virus. J Biomol NMR 21:177-8
Maciejewski, M W; Liu, D; Prasad, R et al. (2000) Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase beta. Correlation with DNA binding and dRP lyase activity. J Mol Biol 296:229-53
Marintchev, A; Robertson, A; Dimitriadis, E K et al. (2000) Domain specific interaction in the XRCC1-DNA polymerase beta complex. Nucleic Acids Res 28:2049-59
Marintchev, A; Maciejewski, M W; Mullen, G P (1999) 1H, 15N, and 13C resonance assignments for the N-terminal 20 kDa domain of the DNA single-strand break repair protein XRCC1. J Biomol NMR 13:393-4
Mullen, G P; Wilson, S H (1997) DNA polymerase beta in abasic site repair: a structurally conserved helix-hairpin-helix motif in lesion detection by base excision repair enzymes. Biochemistry 36:4713-7

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