Assays for the fidelity of DNA polymerases in cell-free extracts of Escherichia coli are complicated by contaminating nucleoside triphosphatases.
In the presence of DNA and a divalent cation, an enzyme activity in cell-free extracts of Escherichia coli readily hydrolyses dATP to dADP. dGTP is degraded to a smaller extent, dCTP and dTTP being hardly affected. The artificial template primers poly(dC) . oligo(dG) and poly(dT) . oligo(dA) are also effective cofactors for this triphosphatase activity. As a consequence, assays measuring the misincorporation, by cell-free extracts, of dATP and dGTP into these defined templates are difficult to interpret, since the triphosphate substrate is being rapidly degraded during the polymerase reaction. A partial characterization of the dATPase activity was performed, demonstrating that the optimal conditions for its activity resemble those commonly used for assaying polymerase activity. Thus in crude extracts both polymerase and dATPase compete for the same substrate. The inclusion of an ATP-generating system in the reaction mixture maintains the levels of deoxynucleoside triphosphates and changes the kinetics of misincorporation of dAMP into poly(dC) . oligo(dG). No reproducible difference in such misincorporation has been found between lysates prepared from tif-1 cells grown at either permissive or restrictive temperature.
D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009708:Nucleotidases / Q000032:analysis*; D013379:Substrate Specificity
D004259:DNA-Directed DNA Polymerase; D009708:Nucleotidases