The field of mutagenesis has benefited from recent important technological advances in cloning and sequencing of DNA. These new methods have been instrumental in establishing the nature and location of mistakes made by DNA polymerases. Now that we know what mutagenic events are occurring in vitro, the question is why do they occur in particular locations on DNA and what determines their frequency of occurrence. The overall objective of this study is aimed at elucidating the kinetic mechanisms of DNA polymerase fidelity. Experiments are proposed in which the kinetics of insertion of single nucleotides, both correct and incorrect, can be determined at specific DNA sites. The data will be used to determine to what extent DNA polymerases utilize base pairing free energy differences or active site geometric contraints to favor the formation of Watson-Crick base pairs over non-complementary base pairs. An analysis of the kinetics of base insertion on unaltered DNA templates will be expanded to include the response of polymerase to a biologically significant noncoding lesion on the DNA template, the apurinic/apyrimidinic site. We will determine how the absence of a coding base influences both the rate and concentration dependence for base insertion opposite the lesion and continuation beyond. The kinetics of incorporating modified nucleotides will also be investigated. Studies with modified bases are fundamentally important for understanding both chemical carcinogenesis and cancer chemotherapy, but further, the homologous series of modified bases that we propose to synthesize will be used to determine how systematic changes in base stacking, ionization, tautomerization, and conformation influence kinetics and fidelity. A version of the Sanger dideoxy DNA sequencing method using modified nucleotides will be used as a rapid and sensitive screening assay to reveal the precise identity of base mispairs and to locate potential mutagenic "hot" and "cold" spots over extended regions of DNA. Nucleotide insertion kinetics at hot and cold spots will then be investigated. In a final series of experiments, we propose to measure the effect of 5-bromouracil, a mutagenic base analogue, on intracellular dNTP pool sizes. Perturbation of dNTP pool sizes is an integral component in understanding base analogue-induced mutagenesis in vivo and this study represents an important continuation of work initiated during the previous grant period.