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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058565-02
Application #
6151233
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Chin, Jean
Project Start
1999-02-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
2
Fiscal Year
2000
Total Cost
$207,815
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Bujalowski, Wlodzimierz; Jezewska, Maria J (2014) Quantitative Thermodynamic Analyses of Spectroscopic Titration Curves. J Mol Struct 1077:40-50
Bujalowski, Wlodek M; Jezewska, Maria J (2012) Fluorescence intensity, anisotropy, and transient dynamic quenching stopped-flow kinetics. Methods Mol Biol 875:105-33
Bujalowski, Wlodek M; Jezewska, Maria J (2012) Using structure-function constraints in FRET studies of large macromolecular complexes. Methods Mol Biol 875:135-64
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz (2011) Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation. J Mol Biol 411:123-42
Jezewska, Maria J; Szymanski, Michal R; Bujalowski, Wlodzimierz (2011) Kinetic mechanism of the ssDNA recognition by the polymerase X from African Swine Fever Virus. Dynamics and energetics of intermediate formations. Biophys Chem 158:9-20
Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Paul J et al. (2011) Full-length Dengue virus RNA-dependent RNA polymerase-RNA/DNA complexes: stoichiometries, intrinsic affinities, cooperativities, base, and conformational specificities. J Biol Chem 286:33095-108
Szymanski, Michal R; Bujalowski, Paul J; Jezewska, Maria J et al. (2011) The N-terminal domain of the Escherichia coli PriA helicase contains both the DNA- and nucleotide-binding sites. Energetics of domain--DNA interactions and allosteric effect of the nucleotide cofactors. Biochemistry 50:9167-83
Bujalowski, Wlodzimierz; Jezewska, Maria J (2011) Macromolecular competition titration method accessing thermodynamics of the unmodified macromolecule-ligand interactions through spectroscopic titrations of fluorescent analogs. Methods Enzymol 488:17-57
Jezewska, Maria J; Szymanski, Michal R; Bujalowski, Wlodzimierz (2011) The primary DNA-binding subsite of the rat pol ?. Energetics of interactions of the 8-kDa domain of the enzyme with the ssDNA. Biophys Chem 156:115-27
Jezewska, Maria J; Szymanski, Michal R; Bujalowski, Wlodzimierz (2011) Interactions of the DNA polymerase X from African Swine Fever Virus with the ssDNA. Properties of the total DNA-binding site and the strong DNA-binding subsite. Biophys Chem 158:26-37

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