ANALYSIS OF MOLECULAR MECHANISMS OF MUTAGENESIS WITH DEFINED COMPONENTS

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Replication and maintenance of the stability of genetic information requires the accurate synthesis of DNA. In animal cells, DNA synthesis is performed by four distinct classes of DNA polymerases, alpha, beta, delta and gamma. Our objective has been to characterize the accuracy of DNA synthesis by each of these enzymes and to analyze the errors committed by each in an attempt to understand how mutation rates are controlled. A major effort this year has been to determine if the high fidelity of chick embryo DNA polymerase gamma results from exonucleolytic proofreading of polymerization errors. This has led to the discovery of a new 3' leads to 5' exonuclease that proofreads so efficiently that this avian polymerase is one of the most accurate DNA polymerases describe to date. This establishes that at least two of the four classes of higher eukaryotic DNA polymerases achieve high fidelity by a proofreading mechanism. We intend to extend these polymerase-gamma studies to mammalian organisms. A second major effort has been to determine the mechanisms by which eukaryotic DNA polymerases discriminate between correct and incorrect nucleotides during polymerization. These efforts have focused on an analysis of the classical miscoding mechanism as well as on base substitution and frameshift errors resulting from misalignment mechanisms. For this purpose, we have just initiated steady state kinetic analyses of misincorporation to determine if the polymerases have active sites that change conformation to reject errors or if error rates are determined primarily by simple nucleotide dissociation rates. Eventually we want to extend these studies to complex enzyme systems that contain additional fidelity components.