The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination.

Zhang H, Rhee C, Bebenek A, Drake JW, Wang J, Konigsberg WH
Biochemistry (2006), Volume 45, Page 2211
PubMed entry Publisher's site

Abstract:

Several variants of RB69 DNA polymerase (RB69 pol) with single-site ...
Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.

Polymerases:

RB69,RB69 D222AD327A/L561A,RB69 Y567F,RB69 D222AL561A,RB69 L561A,RB69 D222AD327A

Topics:

Mutational Analysis, Kinetic Parameters, Structure and Structure/Function, Fidelity, Accessory Proteins/Complexes, Nucleotide Incorporation, Exonuclease Activity, Enzyme Substrate Interactions

One line summary:

Several RB69 mutants were tested for their kinetic parameters as well as their fidelity during DNA replication using an rII assay

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 909uM Reaction: Misincorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 761uM Reaction: Misincorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 4500uM Reaction: Misincorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 725uM Reaction: Misincorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 3363uM Reaction: Misincorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 1715uM Reaction: Misincorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 2085uM Reaction: Misincorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 1986uM Reaction: Misincorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 1391uM Reaction: Misincorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 50uM Reaction: Nucleotide incorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 69uM Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 26uM Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 42uM Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 811uM Reaction: Misincorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 784uM Reaction: Misincorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 1800uM Reaction: Misincorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.004 /second Reaction: Misincorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.011 /second Reaction: Misincorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.77 /second Reaction: Misincorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.041 /second Reaction: Misincorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.001 /second Reaction: Misincorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.09 /second Reaction: Misincorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.013 /second Reaction: Misincorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.14 /second Reaction: Misincorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.015 /second Reaction: Misincorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 220 /second Reaction: Nucleotide incorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 200 /second Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 167 /second Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 240 /second Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.013 /second Reaction: Misincorporation; Substrate: dATP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.003 /second Reaction: Misincorporation; Substrate: dGTP
RB69 The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.08 /second Reaction: Misincorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Frameshift Error Rate 1.2 errors/bp Technique: RB69 rII 131 reversion
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Frameshift Error Rate 4.5 errors/bp Technique: RB69 rII UV232 reversion
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Nucleotide Substitution Rate 2.6 Mutation frequency (relative to WT) Technique: RB69 rII UV256 reversion
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Nucleotide Substitution Rate 4.3 Mutation frequency (relative to WT) Technique: RB69 rII UV375 reversion
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 1467uM Reaction: Misincorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 17uM Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 28uM Reaction: Misincorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 51uM Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 205uM Reaction: Misincorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 155uM Reaction: Misincorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 30uM Reaction: Nucleotide incorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 228uM Reaction: Misincorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 368uM Reaction: Misincorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 310uM Reaction: Misincorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 161uM Reaction: Misincorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 537uM Reaction: Misincorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 251uM Reaction: Misincorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 2010uM Reaction: Misincorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 50uM Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. Kd 507uM Reaction: Misincorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.03 /second Reaction: Misincorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.11 /second Reaction: Misincorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.26 /second Reaction: Misincorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.18 /second Reaction: Misincorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.03 /second Reaction: Misincorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.8 /second Reaction: Misincorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.14 /second Reaction: Misincorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.05 /second Reaction: Misincorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.13 /second Reaction: Misincorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 108 /second Reaction: Nucleotide incorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 89 /second Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 101 /second Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 115 /second Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.03 /second Reaction: Misincorporation; Substrate: dATP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.01 /second Reaction: Misincorporation; Substrate: dGTP
RB69 L561A The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination. kcat 0.75 /second Reaction: Misincorporation; Substrate: dATP
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