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 o f dNTP polymerization catalyzed by DNA polymerase I and the Klenow fra gment was found to be inhibited by the photoaffinity analog of dTTP, w hich acted as a mixed-type inhibitor. In the absence of the reagent bo th forms of the enzyme were rapidly inactivated by UV-irradiation. The substrates (dNTP's and the primer-template complex) protected the enz ymes from UV-induced inactivation. They were not equally active in thi s respect; moreover, in the presence of the primer-template complex th e polymerase activity of DNA polymerase I was higher than in the contr ol Under the conditions of irradiation, the effects of AzdTTP on the t wo 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 o f the analog were covalently bound to each molecule of the enzyme. The template-complementary substrate (dTTP) protected DNA polymerase I fr om both inactivation and modification, while its non-complementary cou nterpart (dCTP) prevented modification but not inactivation. Unlike DN A polymerase I, the Klenow fragment was not inactivated by UV-irradiat ion, AzdTTP failed to modify or significantly affect its polymerase ac tivity. These results suggest that an additional regulatory site respo nsible for dNTP binding may exist within the DNA polymerase I molecule .