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 or against viral infections. At the heart of these processes is the synthesis of the DNA catalyzed by DNA polymerases. Mammalian Polymerase ? (pol ?) and African Swine Fever Virus Polymerase X (pol X) provide outstanding model systems to study the molecular mechanism of the DNA repair polymerase action due to its simplified structures and catalytic repertoires. Because of the fundamental role in human DNA repair and the virus defense against the host reaction to the infection, pol b and pol X are enzymatic systems of a paramount biomedical importance. Mutations and deletions in pol ? have been implicated in several human cancers and genetic diseases including breast, prostate, kidney, lung, colorectal cancers, and Werner syndrome. Mutations in pol X render the virus vulnerable to the DNA-modifying apparatus of the cell, which weakens the effectiveness of the virus infection. In light of the pol ? key role in human DNA repair and the pol X essential role i the effectiveness of viral infection of the mammalian cell, it is of fundamental importance to understand the molecular mechanism by which pol ? and pol X function in performing their activities. Knowledge of mechanistic details of the mechanisms is essential to our understanding of the DNA repair processes in a human cell, the mechanism by which the cell defends itself against diseases, and the mechanism by which the cell fights against viral infections. Studying different steps at the molecular level will provide the necessary knowledge about how to control them. In turn, this knowledge is invaluable for designing rational and efficient therapies for genetic, cancer and viral diseases. The profound and fundamental difference between the replicative and repair polymerases 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. This indicates that DNA and dNTP recognition, which controls fidelity of DNA synthesis, must precede the catalysis. Thus, elucidation of the energetics, dynamics and structure of pol ? - DNA and ASFV pol X - DNA complexes is a prerequisite for understanding the molecular mechanisms of the enzymes, particularly, the efficiency and fidelity of catalysis. The main goal of this project is to elucidate the molecular mechanisms of the recognition of specific DNA structures by pol ? and pol X and their role in DNA synthesis. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of their complexes with DNA substrates and dNTPs in solution using quantitative fluorescence titrations, analytical centrifugation, fluorescence stopped-flow, rapid- quench-flow, fluorescence energy transfer and site-directed mutagenesis techniques.

Public Health Relevance

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 or against viral infections. At the heart of these processes is the synthesis of the DNA catalyzed by DNA polymerases. Elucidation of the polymerase mechanisms is of paramount importance for understanding fundamental processes of the nucleic acid metabolism and why such processes dysfunction in, e.g., cancer and human genetic diseases, and are of fundamental importance in the cell defense against viral infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM058565-14A1
Application #
8439043
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
1999-02-01
Project End
2017-02-28
Budget Start
2013-06-01
Budget End
2014-02-28
Support Year
14
Fiscal Year
2013
Total Cost
$308,271
Indirect Cost
$108,743
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
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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
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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
Andreeva, Iraida E; Szymanski, Michal R; Jezewska, Maria J et al. (2009) Dynamics of the ssDNA recognition by the RepA hexameric helicase of plasmid RSF1010: analyses using fluorescence stopped-flow intensity and anisotropy methods. J Mol Biol 388:751-75
Marcinowicz, Agnieszka; Jezewska, Maria J; Bujalowski, Wlodzimierz (2008) Multiple global conformational states of the hexameric RepA helicase of plasmid RSF1010 with different ssDNA-binding capabilities are induced by different numbers of bound nucleotides. Analytical ultracentrifugation and dynamic light scattering studies. J Mol Biol 375:386-408

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