This research has combined biochemical and cellular investigation on the fidelity of DNA synthesis. The objective is to determine the cellular mechanism that guarantees the accurate copying of the nucleotide sequence of DNA during each cell's division. We also intend to determine whether deficiencies in the accuracy of DNA synthesis are causally associated with mutations and malignancy. Our specific objectives are: (1) to define the accuracy by which DNA polymerases and DNA-replicating complexes copy natural DNA in vitro; (2) to purify cellular factors from animal cells that increase the accuracy of catalysis by DNA polymerases; (3) to study the effect of mutagens and carcinogens on the nature and frequency of misincorporation by DNA polymerases; (4) to analyze the molecular mechanism by which DNA synthesis occurs opposite apurinic sites and to determine the specificity for nucleotide insertion at these sites; and (5) to investigate the relationship between diminution in the fidelity of DNA synthesis and tumor progression. In the past year, we have been able to demonstrate that mutagenesis via apurinic sites is base-specific and involves the insertion of deoxyadenosine into newly synthesized DNA. This specificity provides a marker to quantitate the accumulation of mutations in human DNA that are the result of depurination. The identification of a mutant DNA polymerase-alpha has allowed us to establish a system for transferring the mutant gene for DNA polymerase from one cell to another. This is the first step in the cloning of DNA polymerase-alpha by transfection with recombinant DNA. (I)
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