Mechanism of the ATP effect in the DNA repair synthesis of γ-irradiated Escherichia coli cells

Gamma-irradiation of Escherichia coli cells made permeable to deoxynucleoside triphosphates (dNTP) by toluene induces a repair-type DNA synthesis. As previous studies have shown ATP stimulates this DNA synthesis; we studied the mechanism of the ATP effect by analyzing the kinetics of nucleotide incorporation at various dNTP concentrations. The V values of the DNA repair synthesis rise with increasing dose (0-50 Gy); nonirradiated cells showed a negligible nucleotide incorporation. The apparent Michaelis constant K_M for dNTP in the assay was 83-143 μM and the value was much higher than for a DNA polymerase reaction in vitro. ATP stimulated the DNA synthesis with concomitant decrease of K_M yet unchanged V values. Similar results were obtained with a rec BC strain. We propose that the ATP effect is due to a greater affinity of dNTPs to the DNA polymerase, possibly by a stabilisation of the structural integrity of the complex DNA with repair enzymes. Activation of exonucleases by ATP could be excluded. Addition of NAD to the reaction mixture inhibits the DNA synthesis possibly by activation of ligase which closes the nicks in the DNA strand.