Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured in vitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22 °C to 50 °C, the rate (kpol) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the Kd for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4 × 10−4) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5 × 10−3 at 72 °C which is four-fold higher than that of 37 °C. [Display omitted] ► Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was studied. ► Accuracy of GF was measured at 22 °C, 37 °C, 50 °C using single turnover kinetics. ► kpol increased and Kd changed slightly for a single nucleotide insertion by GF. ► Error frequency of GF remained essentially constant with temperature increase.