The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis

The frequency and specificity of mutations produced during in vitro DNA synthesis of the lacZ alpha gene in M13mp2 DNA by eucaryotic DNA polymerase-alpha (pol-alpha) and DNA polymerase-gamma (pol-gamma) have been determined. Pol-alpha, purified from five different sources, produces mutations resulting in loss of alpha-complementation at a frequency of 0.8-1.6%/single round of gap-filling DNA synthesis. DNA sequence analysis of 420 independent mutants produced by pol-alpha demonstrates three classes of errors. The majority of mutations result from single base substitutions, while single base frameshifts are detected at a lower but substantial frequency. Large deletions are also observed, with a frequency and specificity suggesting that they too are produced by pol-alpha in vitro. In contrast, pol-gamma is more accurate, producing mutants at a frequency of 0.3-0.5%. The specificity of pol-gamma errors is also different, since more than 90% of the mutants result from single base substitutions, while frameshift errors are not observed at a frequency significantly above background. The pol-gamma mutant spectrum also contains deletion mutations (10 of 179 mutants) presumably resulting from aberrant in vitro synthesis. When considered together with previous results using pol-beta (Kunkel, T. A. (1985) J. Biol. Chem. 260, 5787-5796) the relative accuracy of the three classes of purified vertebrate DNA polymerases for base substitutions, frameshifts, and deletions is in the order gamma greater than alpha greater than beta. These data demonstrate a correlation between the accuracy and processivity of DNA polymerization. Thus, the most accurate DNA polymerase (pol-gamma) also incorporates the most nucleotides per association with the primer-template, while the least accurate enzyme (pol-beta) is the least processive. This correlation exists both for base substitution mutations and for single base frameshifts, and is most obvious for minus-one-base frameshifts in runs of pyrimidines. In support of this correlation, increasing the processivity of pol-beta from 1 to 4-6 incorporations per association increases the accuracy of in vitro DNA synthesis by severalfold. The data imply that the processivity of DNA synthesis could be an important factor in controlling the levels of spontaneous and perhaps induced mutations.