The mutational specificity of highly purified and well characterized DNA replication and repair proteins is being examined in in vitro DNA synthesis reactions using biologically active viral DNA templates. Both a forward mutational assay capable of detecting a wide spectrum of errors (base substitutions, frameshifts, deletions, etc.) in M13 mp2 DNA, as well as highly specific reversion assays, are being employed. The work is presently focused on the accuracy of the proteins primarily responsible for the synthesis of new DNA, the DNA polymerases themselves. The accuracy of the three classes of eucaryotic DNA polymerases, established in the forward system, are very different; the mutation frequencies, per round of DNA synthesis in vitro are: polymerase -Gamma, 40 X 10 to the -4; polymerase -Alpha, 80 to 200 X 10 to the -4 and polymerase-Beta, 400 to 800 X 10 to the -4. DNA sequence analysis of over 1000 mutants indicates dramatic and informative differences in the kinds of mutations produced. For example, frameshift mutation frequencies are 300 X 10 to the -4 for PolBeta and less than 1.0 X 10 to the -4 for PolGamma. This correlates with the "Processivity" of these enzymes. Comparably large differences in specific base substitution errors are also observed. These experiments are intended to provide detailed information on the parameters of protein-nucleic acid interactions which are important in determining accuracy. The studies are being expanded to employ prokaryotic DNA polymerases capable of proofreading, as well as additional "accessory" proteins from prokaryotic and eukaryotic systems.