The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DMA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DMA. The question of polymerase accuracy has important health implications because the errors made by DMA polymerases can result in mutations leading to human disease. Moreover, DMA polymerases are frequently targeted in chemotherapeutic and antiviral strategies, as well as being important in a variety of diagnostic biotechnology applications, so an understanding of their reaction mechanisms is crucial. Our investigations focus on two model DMA polymerases that have contrasting enzymatic properties: the highly accurate DMA polymerase I (Klenow fragment) of E. coli, and the much less accurate Dbh bypass polymerase from the archaeon S. solfataricus. Structural data are available for both these enzymes and several close homologues, and serve as the basis for many of the planned experiments. Moreover, because the important features of the polymerase active site and reaction mechanism are conserved throughout the polymerase family, the results obtained with these simple model systems will have much wider relevance. A major priority will be the investigation of noncovalent steps in the polymerase reaction pathway because these conformational transitions are likely to be involved in distinguishing between correctly paired substrates and the mispairs that result in polymerase errors. We will use fluorescence assays in combination with rapid single-turnover kinetics to investigate the rates of conformational changes and the effect on the reaction pathway of mispaired substrates, active site mutations and damaged DMA. DMA damage and errors in DMA synthesis can cause mutations that lead to diseases such as cancer, making it important to understand how DMA polymerases function to avoid these outcomes. Antiviral drugs frequently target viral polymerases so research into polymerase structures and mechanism is relevant in designing effective drugs and understanding how they work. DNA polymerases are also a crucial part of many of the diagnostic tools used in modern medicine and will be pivotal in the development of new DNA sequencing technologies for diagnostic purposes.
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