[Features of interaction of Escherichia coli DNA polymerase I and its Klenow fragment with dTTP gamma-p-azidoanilide]

Biokhimiia. 1993 Feb;58(2):224-33.
[Article in Russian]


gamma-p-Azidoanilidate of dTTP was used to study the photoaffinity modification of DNA polymerase I and Klenow fragment. The analog was found to be a mixed-type inhibitor with respect to dTTP of the polymerization reaction catalyzed by DNA polymerase I and Klenow fragment. In the absence of the reagent both UV-irradiated enzymes were rapidly inactivated. Substrates (dNTP and template-primer) protected the enzymes from inactivation by UV-light with different efficiency. In the presence of the template-primer UV-irradiation induced activation of DNA polymerase I. The effect of the analog on both enzyme forms under irradiation is different. At concentration of 10(-5)M gamma-p-anilidate of dTTP accelerated the activation of DNA polymerase I initiated by UV-irradiation and at 10(-4)M concentration it inactivated the enzyme by 20-25%. Under such conditions one enzyme molecule covalently bound two molecules of the analog. While the template-complementary substrate (dTTP) protected DNA polymerase I both from inactivation and modification, the non-complementary one (dCTP) worked only against modification. In contrast to DNA polymerase I Klenow fragment was not inactivated when exposed to UV-irradiation and gamma-p-anilidate of dTTP neither modified the protein nor exerted any significant effect on its polymerization activity. The data accumulated suggest the presence on the DNA polymerase I molecule of a regulatory region providing additional dNTP binding sites.

MeSH terms

  • Azides / metabolism*
  • DNA Polymerase I / antagonists & inhibitors
  • DNA Polymerase I / metabolism*
  • DNA Polymerase I / radiation effects
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology*
  • Substrate Specificity
  • Thymine Nucleotides / metabolism*
  • Ultraviolet Rays


  • Azides
  • Thymine Nucleotides
  • dTTP gamma-4-azidoanilide
  • DNA Polymerase I