Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases.

Abstract:

For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase beta-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed in in vitro reactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the beta hairpin, to the exonuclease active site, or to both. We propose from these studies that the beta hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3' end of the primer strand following strand separation.

Polymerases:

Topics:

Mutational Analysis, Other Enzymatic Activities, Structure and Structure/Function, Fidelity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 Y254S Subuddhi U2008 Nucleotide Substitution Rate 36 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 Y254Q Subuddhi U2008 Nucleotide Substitution Rate 47 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 Y254K Subuddhi U2008 Nucleotide Substitution Rate 77 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 Y254L Subuddhi U2008 Nucleotide Substitution Rate 7 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 Y254G Subuddhi U2008 Nucleotide Substitution Rate 72 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 G255S Subuddhi U2008 Nucleotide Substitution Rate 113 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 G255A Subuddhi U2008 Nucleotide Substitution Rate 107 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 S256Q Subuddhi U2008 Nucleotide Substitution Rate 1 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 S256Y Subuddhi U2008 Nucleotide Substitution Rate 1 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 S256K Subuddhi U2008 Nucleotide Substitution Rate 11 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 S256L Subuddhi U2008 Nucleotide Substitution Rate 15 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 K257S Subuddhi U2008 Nucleotide Substitution Rate 1 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 K257Q Subuddhi U2008 Nucleotide Substitution Rate 4 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 K257Y Subuddhi U2008 Nucleotide Substitution Rate 3 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 K257L Subuddhi U2008 Nucleotide Substitution Rate 3 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
T4 K257G Subuddhi U2008 Nucleotide Substitution Rate 6 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay
RB69 G258S Subuddhi U2008 3-5' Exonuclease (proofreading) No
RB69 beta hairpin- Subuddhi U2008 3-5' Exonuclease (proofreading) No
RB69 D222AD327A Subuddhi U2008 3-5' Exonuclease (proofreading) No
RB69 G258SD222A/D237A Subuddhi U2008 3-5' Exonuclease (proofreading) No
RB69 D222AD327A beta hairpin- Subuddhi U2008 3-5' Exonuclease (proofreading) No
RB69 D222AD327A beta hairpin- Subuddhi U2008 5-3' Exonuclease Unspecified
T4 S256G Subuddhi U2008 Nucleotide Substitution Rate 34 Mutation frequency (relative to WT) Technique: T4 rII 199oc reversion assay

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