This work is devoted to the development of synthetic reagents for use as tools in the study of the structure and function of replicative DNA polymerases. A long term goal is to define amino acids in the active site region of DNA polymerases and the mechanism by which amino acids in the site effect incorporation of dNTPs at a primer terminus. A model enzyme for these studies is bacteriophage T4 DNA polymerase. A second goal is to clarify the relationship between eukaryotic DNA polymerases alpha, delta, epsilon, and to provide reagents to help define their roles in the substeps of DNA replication in vivo. This work will build upon previous results indicating that the synthetic nucleotides, N2-(p-n-butylphenyl) dGTP (BuPdGTP) and 2-(p-n-butylanilino (BuAdATP), are potent and selective inhibitors of DNA polymerase alpha and T4 DNA polymerase, and that carbonyldiphosphonate (COMDP) is a selective inhibitor of DNA polymerase de a.
Four specific aims are proposed: development and characterization of selective inhibitors of calf thymus and HeLa cell DNA polymerases delta and epsilon; dissection of the mechanism by which the potent pol alpha inhibitor, BuPdGTP, inhibits and is incorporated by bacteriophage T4 DNA polymerase; synthesis and use of reactive, active site-directed nucleotides to label and dissect the active sites of DNA polymerases alpha and delta and T4 DNA polymerase, and; synthesis and demonstration of stable dNTP and primer analogs for use in X-ray crystallographic study of T4 DNA polymerase. Methods of organic synthesis are used to prepare synthetic nucleotides and phosphonates as inhibitors, substrates and potential irreversible labels for selected DNA polymerases. Ion exchange and reverse phase chromatography and multinuclear NMR spectroscopy are techniques used to support the synthetic work. DNA polymerase isolation and purification, enzyme assay by radioactive substrate incorporation and by polyacrylamide gel electrophoretic (PAGE) analysis of primer-extension reactions, and isolation by HPLC of modified primers will be used for biochemical work. Experiments involving protein degradation and chemical and spectroscopic analysis of labelled amino acids or peptides will also be employed.
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