IDENTIFICATION OF A TRANSIENT EXCISION INTERMEDIATE AT THE CROSSROADSBETWEEN DNA-POLYMERASE EXTENSION AND PROOFREADING PATHWAYS

DNA polymerases achieve accurate DNA replication through a delicate ba lance between primer elongation and proofreading, While insufficient p roofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may preven t completion of DNA synthesis, The transition between polymerization a nd proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is sig nificantly reduced by the presence of an incorrect base pair at the pr imer-terminus. We have used mutational analysis, coupled with a sensit ive, fluorescence-based assay to characterize intermediate steps in th e proofreading pathway, A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at t he 3' terminus with the base analog: 2-aminopurine. Formation of the f luorescent complex appears to be a rate-determining step in the proofr eading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain, Although thes e mutant DNA polymerases are proficient in hydrolysis, their reduced a bility to form the fluorescent complex imposes a higher kinetic barrie r, As a consequence, the mutant DNA polymerases proofread less frequen tly, resulting in more DNA replication errors.