SPECIFIC EFFECTS OF DTTP GAMMA-P-AZIDOANILIDE ON ESCHERICHIA-COLI DNA-POLYMERASE-I AND ITS KLENOW FRAGMENT

Photoaffinity modification of DNA polymerase I and its Klenow fragment by dTTP gamma-p-azidoanilide (AzdTTP) has been studied The reaction of dNTP polymerization catalyzed by DNA polymerase I and the Klenow fragment was found to be inhibited by the photoaffinity analog of dTTP, which acted as a mixed-type inhibitor. In the absence of the reagent both forms of the enzyme were rapidly inactivated by UV-irradiation. The substrates (dNTP's and the primer-template complex) protected the enzymes from UV-induced inactivation. They were not equally active in this respect; moreover, in the presence of the primer-template complex the polymerase activity of DNA polymerase I was higher than in the control Under the conditions of irradiation, the effects of AzdTTP on the two forms of the enzyme were different. At 10(-5) M, AzdTTP facilitated UV-induced activation of DNA polymerase I, at 10(-4) M the enzyme was inactivated by 20 to 25%. Under the latter conditions two molecules of the analog were covalently bound to each molecule of the enzyme. The template-complementary substrate (dTTP) protected DNA polymerase I from both inactivation and modification, while its non-complementary counterpart (dCTP) prevented modification but not inactivation. Unlike DNA polymerase I, the Klenow fragment was not inactivated by UV-irradiation, AzdTTP failed to modify or significantly affect its polymerase activity. These results suggest that an additional regulatory site responsible for dNTP binding may exist within the DNA polymerase I molecule.