Dynamics of bacteriophage T4 DNA polymerase function: identification of amino acid residues that affect switching between polymerase and 3' --> 5' exonuclease activities.

Abstract:

Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides. Since polymerase and 3'--> 5' exonuclease activities appear to reside in spatially distinct active centers, there must be some mechanism for coordinating replication with proofreading and for transferring DNA between the two active centers. We have designed a genetic selection scheme to isolate bacteriophage T4 mutant DNA polymerases that are defective in "switching" between polymerase and exonuclease activities. Amino acid residues that affected active-site-switching were identified in four regions of the T4 DNA polymerase: two regions in the proposed exonuclease domain. Representative mutant DNA polymerases from each region were purified for biochemical studies. We propose that amino acid substitutions identified by mutational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase and exonuclease active centers.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Structure and Structure/Function, Fidelity, Exonuclease Activity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 I50L Stocki SA1995 Nucleotide Substitution Rate 100 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 64[KD]uplication Stocki SA1995 Nucleotide Substitution Rate 50 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 N84K Stocki SA1995 Nucleotide Substitution Rate 100 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 G255S Stocki SA1995 Nucleotide Substitution Rate 58 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 271[L]uplication Stocki SA1995 Nucleotide Substitution Rate 74 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 V355A Stocki SA1995 Nucleotide Substitution Rate 5 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 Q380K Stocki SA1995 Nucleotide Substitution Rate 5 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 E395K Stocki SA1995 Nucleotide Substitution Rate 14 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 L412M Stocki SA1995 Nucleotide Substitution Rate 9 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 E743K Stocki SA1995 Nucleotide Substitution Rate 11 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 S756P Stocki SA1995 Nucleotide Substitution Rate 13 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 L771F Stocki SA1995 Nucleotide Substitution Rate 0.3 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 L771H Stocki SA1995 Nucleotide Substitution Rate 8 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 [L771+D] Stocki SA1995 Nucleotide Substitution Rate 29 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 [L771+V] Stocki SA1995 Nucleotide Substitution Rate 22 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay

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