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

Abstract:

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:

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 Zhang H2006 Kd 909uM Reaction: Misincorporation; Substrate: dATP
RB69 Zhang H2006 Kd 761uM Reaction: Misincorporation; Substrate: dGTP
RB69 Zhang H2006 Kd 4500uM Reaction: Misincorporation; Substrate: dCTP
RB69 Zhang H2006 Kd 725uM Reaction: Misincorporation; Substrate: dGTP
RB69 Zhang H2006 Kd 3363uM Reaction: Misincorporation; Substrate: dCTP
RB69 Zhang H2006 Kd 1715uM Reaction: Misincorporation; Substrate: dTTP
RB69 Zhang H2006 Kd 2085uM Reaction: Misincorporation; Substrate: dCTP
RB69 Zhang H2006 Kd 1986uM Reaction: Misincorporation; Substrate: dTTP
RB69 Zhang H2006 Kd 1391uM Reaction: Misincorporation; Substrate: dTTP
RB69 Zhang H2006 Kd 50uM Reaction: Nucleotide incorporation; Substrate: dATP
RB69 Zhang H2006 Kd 69uM Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 Zhang H2006 Kd 26uM Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 Zhang H2006 Kd 42uM Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 Zhang H2006 Kd 811uM Reaction: Misincorporation; Substrate: dATP
RB69 Zhang H2006 Kd 784uM Reaction: Misincorporation; Substrate: dGTP
RB69 Zhang H2006 Kd 1800uM Reaction: Misincorporation; Substrate: dATP
RB69 Zhang H2006 kcat 0.004 /second Reaction: Misincorporation; Substrate: dATP
RB69 Zhang H2006 kcat 0.011 /second Reaction: Misincorporation; Substrate: dGTP
RB69 Zhang H2006 kcat 0.77 /second Reaction: Misincorporation; Substrate: dCTP
RB69 Zhang H2006 kcat 0.041 /second Reaction: Misincorporation; Substrate: dGTP
RB69 Zhang H2006 kcat 0.001 /second Reaction: Misincorporation; Substrate: dCTP
RB69 Zhang H2006 kcat 0.09 /second Reaction: Misincorporation; Substrate: dTTP
RB69 Zhang H2006 kcat 0.013 /second Reaction: Misincorporation; Substrate: dCTP
RB69 Zhang H2006 kcat 0.14 /second Reaction: Misincorporation; Substrate: dTTP
RB69 Zhang H2006 kcat 0.015 /second Reaction: Misincorporation; Substrate: dTTP
RB69 Zhang H2006 kcat 220 /second Reaction: Nucleotide incorporation; Substrate: dATP
RB69 Zhang H2006 kcat 200 /second Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 Zhang H2006 kcat 167 /second Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 Zhang H2006 kcat 240 /second Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 Zhang H2006 kcat 0.013 /second Reaction: Misincorporation; Substrate: dATP
RB69 Zhang H2006 kcat 0.003 /second Reaction: Misincorporation; Substrate: dGTP
RB69 Zhang H2006 kcat 0.08 /second Reaction: Misincorporation; Substrate: dATP
RB69 L561A Zhang H2006 Frameshift Error Rate 1.2 errors/bp Technique: RB69 rII 131 reversion
RB69 L561A Zhang H2006 Frameshift Error Rate 4.5 errors/bp Technique: RB69 rII UV232 reversion
RB69 L561A Zhang H2006 Nucleotide Substitution Rate 2.6 Mutation frequency (relative to WT) Technique: RB69 rII UV256 reversion
RB69 L561A Zhang H2006 Nucleotide Substitution Rate 4.3 Mutation frequency (relative to WT) Technique: RB69 rII UV375 reversion
RB69 L561A Zhang H2006 Kd 1467uM Reaction: Misincorporation; Substrate: dCTP
RB69 L561A Zhang H2006 Kd 17uM Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 L561A Zhang H2006 Kd 28uM Reaction: Misincorporation; Substrate: dTTP
RB69 L561A Zhang H2006 Kd 51uM Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 L561A Zhang H2006 Kd 205uM Reaction: Misincorporation; Substrate: dTTP
RB69 L561A Zhang H2006 Kd 155uM Reaction: Misincorporation; Substrate: dATP
RB69 L561A Zhang H2006 Kd 30uM Reaction: Nucleotide incorporation; Substrate: dATP
RB69 L561A Zhang H2006 Kd 228uM Reaction: Misincorporation; Substrate: dGTP
RB69 L561A Zhang H2006 Kd 368uM Reaction: Misincorporation; Substrate: dATP
RB69 L561A Zhang H2006 Kd 310uM Reaction: Misincorporation; Substrate: dATP
RB69 L561A Zhang H2006 Kd 161uM Reaction: Misincorporation; Substrate: dGTP
RB69 L561A Zhang H2006 Kd 537uM Reaction: Misincorporation; Substrate: dCTP
RB69 L561A Zhang H2006 Kd 251uM Reaction: Misincorporation; Substrate: dGTP
RB69 L561A Zhang H2006 Kd 2010uM Reaction: Misincorporation; Substrate: dCTP
RB69 L561A Zhang H2006 Kd 50uM Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 L561A Zhang H2006 Kd 507uM Reaction: Misincorporation; Substrate: dTTP
RB69 L561A Zhang H2006 kcat 0.03 /second Reaction: Misincorporation; Substrate: dATP
RB69 L561A Zhang H2006 kcat 0.11 /second Reaction: Misincorporation; Substrate: dGTP
RB69 L561A Zhang H2006 kcat 0.26 /second Reaction: Misincorporation; Substrate: dCTP
RB69 L561A Zhang H2006 kcat 0.18 /second Reaction: Misincorporation; Substrate: dGTP
RB69 L561A Zhang H2006 kcat 0.03 /second Reaction: Misincorporation; Substrate: dCTP
RB69 L561A Zhang H2006 kcat 0.8 /second Reaction: Misincorporation; Substrate: dTTP
RB69 L561A Zhang H2006 kcat 0.14 /second Reaction: Misincorporation; Substrate: dCTP
RB69 L561A Zhang H2006 kcat 0.05 /second Reaction: Misincorporation; Substrate: dTTP
RB69 L561A Zhang H2006 kcat 0.13 /second Reaction: Misincorporation; Substrate: dTTP
RB69 L561A Zhang H2006 kcat 108 /second Reaction: Nucleotide incorporation; Substrate: dATP
RB69 L561A Zhang H2006 kcat 89 /second Reaction: Nucleotide incorporation; Substrate: dGTP
RB69 L561A Zhang H2006 kcat 101 /second Reaction: Nucleotide incorporation; Substrate: dCTP
RB69 L561A Zhang H2006 kcat 115 /second Reaction: Nucleotide incorporation; Substrate: dTTP
RB69 L561A Zhang H2006 kcat 0.03 /second Reaction: Misincorporation; Substrate: dATP
RB69 L561A Zhang H2006 kcat 0.01 /second Reaction: Misincorporation; Substrate: dGTP
RB69 L561A Zhang H2006 kcat 0.75 /second Reaction: Misincorporation; Substrate: dATP

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.