Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways.

Abstract:

DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2-aminopurine. Formation of the fluorescent complex appears to be a rate-determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.

Polymerases:

Topics:

Mutational Analysis, Biotech Applications, Kinetic Parameters, Structure and Structure/Function, Fidelity, Nucleotide Incorporation, Exonuclease Activity, Enzyme Substrate Interactions, Methods

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D112AE114A Baker RP1998 Nucleotide Substitution Rate 300 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 G255S Baker RP1998 Nucleotide Substitution Rate 58 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D131N Baker RP1998 Nucleotide Substitution Rate 15 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 D131G Baker RP1998 Nucleotide Substitution Rate 250 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay

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