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 (3 (pol p) 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 P and pol X are enzymatic systems of a paramount biomedical importance. Mutations and deletions in pol p 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 progress of the virus infection. In light of the pol (3 key role in human DNA repair and the pol X essential role in the effectiveness of viral infection of the mammalian cell, it is of fundamental importance to understand the molecular mechanism by which pol p 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 p - 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 p 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.
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