MOTIF-A OF BACTERIOPHAGE-T4 DNA-POLYMERASE - ROLE IN PRIMER EXTENSIONAND DNA-REPLICATION FIDELITY - ISOLATION OF NEW ANTIMUTATOR AND MUTATOR DNA-POLYMERASES

Polymerases in general share only a few regions of amino acid similari ty. One of the most conserved regions, called motif A, has the sequenc e DXXSLYPSII or a similar sequence in many eukaryotic and viral DNA po lymerases and in bacteriophage T4 DNA polymerase. We designed genetic techniques to isolate mutant T4 DNA polymerases with amino acid substi tutions in this highly conserved motif. The mutant DNA polymerases dif fered from wild type T4 DNA polymerase in several ways. For one mutant DNA polymerase, the pyrophosphate analog, phosphonoacetic acid, was a potent inhibitor of DNA replication, and this mutant DNA polymerase r eplicated DNA with reduced fidelity. Another mutant DNA polymerase rep licated DNA with increased accuracy, but this mutant DNA polymerase wa s less processive in primer extension reactions, and DNA replication r equired high concentrations of deoxynucleoside triphosphates. We provi de evidence that indicates that all of these changes to DNA polymerase function are due to differences in how the mutant DNA polymerases par tition between states active for DNA replication or exonucleolytic pro ofreading. These studies also provide further support for the hypothes is that the accuracy of DNA replication observed for DNA polymerases d epends on the interplay between polymerase and 3' --> 5' exonuclease a ctivities (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. B iol. Chem. 247, 7116-7122).