MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

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DESCRIPTION: The long term goal of the proposed research is to understand the molecular events involved during template dependent DNA synthesis. The Klenow fragment of the E. coli DNA polymerase I will be utilized as a prototype model system, since extensive characterization of the mechanistic and structural properties of this enzyme have been carried out. It is the first DNA polymerase whose crystal structure has been solved. The domains of amino acid residues in this enzyme which participate in the binding of substrates and/or contribute to the catalytic process of phosphodiester bond formation will be defined. For this purpose, both affinity labeling and site-directed mutagenesis approaches will be implemented. In addition, availability of the crystal coordinates of the Klenow fragment has permitted model building of the complete 3-dimensional structure with side chains using molecular simulation and modeling programs. The model built structure serves as an excellent guide in planning as well as in the interpretation of results. This 3D structure may also suggest amino acid residues which are likely to participate in specific functions providing a good experimental target for mutagenesis. Results obtained with mutant enzymes would either confirm the prediction or would suggest modifications to the existing structural models and their functional implications. As a result of these efforts the investigator hopes to identify a) functionally important amino acid residues in pol I, b) clarify the role they play including interactions with substrates, c) deduce functional domains contributed by different amino acids, and d) clarify the mechanism of template-dependent substrate selection and the mechanisms of its addition onto primer terminus.