THE PROOFREADING PATHWAY OF BACTERIOPHAGE-T4 DNA-POLYMERASE

The base analog, 2-aminopurine (2AP), was used as a fluorescent report er of the biochemical steps in the proofreading pathway catalyzed by b acteriophage T4 DNA polymerase, ''Mutator'' DNA polymerases that are d efective in different steps in the exonucleolytic proof-reading pathwa y were studied so that transient changes in fluorescence intensity cou ld be equated with specific reaction steps. The G255S- and D131N-DNA p olymerases can hydrolyze DNA, the final step in the proofreading pathw ay, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrol ysis-defective D112A/E114A-DNA polymerase was also examined. Fluoresce nt enzyme-DNA complexes were preformed in the absence of Mg2+, and the n rapid mixing, stopped-flow techniques were used to determine the fat e of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathwa y. These studies are consistent with a proofreading pathway in which t he protein loop structure that contains residue Gly(255) functions in strand separation and transfer of the primer strand from the polymeras e active center to form a preexonuclease complex. Residue Asp(131) act s at a later step in formation of the preexonuclease complex.