Kinetics of error generation in homologous B-family DNA polymerases.

Abstract:

The kinetics of forming a proper Watson-Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all approximately 2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase.

Polymerases:

Topics:

Mutational Analysis, Kinetic Parameters, Nucleotide Analogs / Template Lesions, Structure and Structure/Function, Fidelity, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
RB69 D114AE116A Hogg M2006 Kd 47uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
RB69 D114AE116A Hogg M2006 Kd 832uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
RB69 D114AE116A Hogg M2006 kcat 270 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
RB69 D114AE116A Hogg M2006 kcat 0.43 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D112AE114A Hogg M2006 Kd 11uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
T4 D112AE114A Hogg M2006 Kd 915uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D112AE114A Hogg M2006 kcat 361 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
T4 D112AE114A Hogg M2006 kcat 3.24 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
RB69 D327A Hogg M2006 Kd 47uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
RB69 D327A Hogg M2006 Kd 1210uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D324A Hogg M2006 Kd 18uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
T4 D324A Hogg M2006 Kd 897uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D324A Hogg M2006 kcat 180 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
T4 D324A Hogg M2006 kcat 2.13 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
RB69 D327A Hogg M2006 kcat 224 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
RB69 D327A Hogg M2006 kcat 0.53 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D219A Hogg M2006 Kd 13uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
T4 D219A Hogg M2006 Kd 934uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D219A Hogg M2006 kcat 402 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
T4 D219A Hogg M2006 kcat 3.36 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
T4 D219A Hogg M2006 kcat 0.56 /second Reaction: Misincorporation; Substrate: dATP; Technique: Multiple Turnover (across template C)
T4 D219A Hogg M2006 kcat 0.18 /second Reaction: Misincorporation; Substrate: dTTP; Technique: Multiple Turnover (across template T)
RB69 D222A Hogg M2006 Kd 42uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
RB69 D222A Hogg M2006 Kd 1767uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
RB69 D222A Hogg M2006 kcat 320 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template T)
RB69 D222A Hogg M2006 kcat 0.97 /second Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Single Turnover (across template F)
RB69 D222A Hogg M2006 kcat 0.43 /second Reaction: Misincorporation; Substrate: dATP; Technique: Multiple Turnover (across template C)
RB69 D222A Hogg M2006 kcat 0.04 /second Reaction: Misincorporation; Substrate: dTTP; Technique: Multiple Turnover (across template T)

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