[Features of interaction of Escherichia coli DNA polymerase I and its Klenow fragment with dTTP gamma-p-azidoanilide].
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.
Kudriashova
N V
NV
Shamanina
M Iu
MIu
Godovikova
T S
TS
Anan'ko
E A
EA
Akhmadieva
F F
FF
Romashchenko
A G
AG
D001386:Azides / Q000378:metabolism*; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism*; D014466:Ultraviolet Rays
D001386:Azides; D013942:Thymine Nucleotides; C081150:dTTP gamma-4-azidoanilide; D004256:DNA Polymerase I
Osobennosti vzaimodeĭstviia DNK-polimerazy I Escherichia coli i ee bol'shogo fragmenta s gamma-p-azidoanilidom dTTP.