[Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs]. / Vzaimodeistvie dNTP-sviazyvaiushchikh uchastkov DNK-polimerazy alpha cheloveka i fragmenta Klenova DNK-polimerazy I iz Escherichia coli s nukleotidami, pirofosfatom i ikh analogami.

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.

[The effect of various substituents, joined through the 5'- and 3'-ends of the primer, initiating properties during the polymerization reaction, catalyzed by AMV-revertase]. / Vliianie razlichnykh zamestitelei, prisoedinennykh po 5'- i 3'-kontsampraimera, na ego initsiiruiushchie svoistva v reaktsii polimerizatsii, kataliziruemoi AMV-revertazoi.

We investigated the interaction of AMV reverse transcriptase and Klenow fragment with oligonucleotide derivatives carrying different 3'- or 5'-terminal reactive groups. It was shown that the attachment of phenazinium, ethidium, and daunomycin residues to the 5'-terminal phosphate stabilized the enzyme template primer complexes, while cholesterol and hemin residues generally decreased their stability. The increased stability in solution correlated to a certain extent with the increase in affinity of the modified primers to the enzyme template complex. Coupling of bulky R residues to the primers had a weak effect on the maximal rate of primer conversion, which is likely to be a result of the lack of strong contacts between the substituents and the enzyme, and steric obstacles hindering translocation of the primer enzyme complex. We analyzed the inhibitory effect of 23 oligonucleotide derivatives (both complementary and noncomplementary to the template) with modified 3'- and 5'-ends, and revealed several analogs inhibiting polymerization catalyzed by AMV reverse transcriptase by 70-100% at 0.1-1 microM concentrations of the reagents.