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. Having found that the high fidelity of the mitochondrial replicative DNA polymerase gamma from chick results from exonucleolytic proofreading, we searched for and found similar activity in mammalian gamma polymerases from two sources. This generalizes the discovery of proofreading activity associated with this class of polymerase, and establishes that two of the four classes of higher eukaryotic DNA polymerases achieve high fidelity by a proofreading mechanism. to determine the mechanisms by which eukaryotic DNA polymerases discriminate between correct and incorrect nucleotides during polymerization, we have analyzed base substitutions produced by DNA polymerase-beta by both classical miscoding and transient misalignment mechanisms, using steady state enzyme kinetic analyses. A major focus during the past year has been a continuing examination of the fidelity of the two putative replicative DNA polymerase, alpha and delta. We have established that the four-subunit DNA polymerase alpha-DNA primase complex purified from four different sources by immunoaffinity chromatography is no more accurate than the conventionally purified polymerase. Studies to examine the relationship between the processivity and fidelity of polymerization have begun. Unlike Pol alpha, DNA polymerase Delta is highly accurate, which is at least in part due to exonucleolytic proofreading. Two forms of the enzyme are being studied, a PCNA-stimulable and a PCNA-independent form. The latter enzyme is highly accurate for base substitution errors and is currently being tested in a forward mutation assay to examine error specificity. The former enzyme is being similarly tested, and the involvement of PCNA and its contribution to fidelity, if any, are under investigation.