BASE MISCODING AND STRAND MISALIGNMENT ERRORS BY MUTATOR KLENOW POLYMERASES WITH AMINO-ACID SUBSTITUTIONS AT TYROSINE-766 IN THE O-HELIX OFTHE FINGERS SUBDOMAIN

A mutant derivative of Klenow fragment DNA polymerase containing serin e substituted for tyrosine at residue 766 has been shown by kinetic an alysis to have an increased misinsertion rate relative to wild-type Kl enow fragment, but a decreased rate of extension from the resulting mi spairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochem istry 30, 804-813). In the present study we use an M13mp2-based fideli ty assay to study the error specificity of this mutator polymerase. De spite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error fates, The magnitude of the mutator effect is mispair-specific, from no effec t for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are rema rkably consistent with the earlier kinetic analysis of misinsertion, d emonstrating that either approach can be used to identify and characte rize mutator polymerases, Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base delet ions and a 276-base deletion between a direct repeat sequence, However , neither mutant polymerase has an increased error rate for single-bas e frameshifts in repetitive sequences, This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indic ate that the nucleotide misinsertion and strand-slippage mechanisms fo r polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar sub domain structures.