[Photoactivated analogues of the initiating substrates of RNA polymerase II based on arylazide derivatives of NTP gamma-amidophosphate: synthesis and chemical and photochemical reactions of functional groups].

Photoactivatable derivatives Ar-NH-(CH2)n-NHpppB (where Ar = p-azidophenyl (A1), 5-azido-2-nitrobenzoyl (A2), or 4-azido-2,3,5,6-tetrafluorobenzoyl (A3) group; B = Ado or Guo; n = 2, 3, or 4) were synthesized. The phosphoroamidate bond stability was found to depend on the structure of both the heterocyclic and the photoactivatable groups. The derivative with A3, Ado, and n=3 is hydrolyzed with regeneration of aryl azide and ATP, whereas the other derivatives are stable in aqueous solutions. The photoanalogues with A1 and A2, B = Ado, and n = 2 or 4 were found to behave as initiating substrates toward the RNA polymerase II from Saccharomyces cerevisiae under the conditions of specific transcription initiation and control of the adenovirus late promoter. The photolysis of N-(4-azidophenyl)-1,4-diaminobutane and N-(5-azido-2-nitrobenzoyl)-1,3-diaminopropane, two functional fragments of the photoaffinity reagents, in aqueous solutions was established to result in the formation of p-benzoquinone diimine and p-nitro-N-arylhydroxylamine derivatives, respectively. The arylhydroxylamine derivatives undergo a number of transformations in aqueous solution leading to nitroso derivatives. We concluded that it is these nitroso derivatives (products of nitrene transformation, rather than the nitrene itself) that may modify proteins with reagents containing p-nitrophenylazide fragment.

[Features of interaction of Escherichia coli DNA polymerase I and its Klenow fragment with dTTP gamma-p-azidoanilide]. / Osobennosti vzaimodeistviia DNK-polimerazy I Escherichia coli i ee bol'shogo fragmenta s gamma-p-azidoanilidom dTTP.

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.

[Affinity modification of Escherichia coli Dna-polymerase I by 4-(N-2-chloroethyl-N-methylamino)-benzyl-gamma-amides of dTTP and dATP]. / Affinnaia modifikatsiia DNK-polimerazy I iz Escherichia coli 4-(N-2-chloroéthyl-N-methylamino)-benzyl-gamma-amidami dTTP i dATP.

The interaction of dTTP and dATP gamma-4-(N-2-chloroethyl-N-methylamino) benzylamidates with E. coli DNA-polymerase I were studied. dTTP and dATP gamma-4-(N-2-hydroxyethyl-N-methylamino) benzylamidates act as competitive inhibitors of DNA-polymerase I. Ki values for gamma-analogues of dTTP and dATP have been determined. Reactive dTTP and dATP derivatives are shown to be affinity reagent for this enzyme.