Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates.

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.

[Recognition of T-T cyclobutane pyrimidine dimers in the form of primers by DNA polymerases]. / Uznavanie DNK-polimerazami T-T tsiklobutanovykh pirimidinovykh dimerov v sostave praimerov.

A study was made of the efficiency of primer conversion catalyzed by human placenta DNA polymerase on the length of primer. The dependence of -log Kd and Vmax on the number of mononucleotide units (n) of primer were shown to be linear up to n <--> 10. Each mononucleotide unit of the primer enhanced its affinity by a factor of 2.3 and the maximal polymerization rate by a factor of 1.3. For the first time estimation was made for the contribution of replication enzymes: human placenta DNA polymerase and E. coli DNA polymerase I (as well as AMV reverse transcriptase) to a decrease in the amount of T-T dimers in DNA emerging after UV- or gamma-irradiation. The efficiency of elongation of the d(pT)10 primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10 primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

[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.

5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1.

The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.

A comparison of the initiating abilities of ribo- and deoxyriboprimers in DNA polymerization catalyzed by AMV reverse transcriptase.

The difference in optimal conditions for DNA polymerization catalyzed by AMV reverse transcriptase on poly(A) and poly(dA) templates with d(pT)10 and (pU)10 primers has been found. A comparison of the initiating abilities of d(pT)10 and (pU)10 primers under optimal conditions for various template.primer complexes has been made. The best template.primer complex was poly(A).d(pT)10 and the worst was poly(A).(pU)10. The lengthening of d(pT)n primers by a mononucleotide unit (n = 2-10) increases their affinity by a factor of about 2 and 3 in the case of poly(dA) and poly(A) templates, respectively. The affinities of d(pT) to the enzyme does not change with the primer length.

[The effect of bases noncomplementary to the template on the effectiveness of interaction between primers and avian myeloblastosis virus reverse transcriptase]. / Vliianie ne komplementarnykh matritse osnovanii na éffektivnost' vzaimodeistviia praimerov s obratnoi transkriptazoi virusa mieloblastoza ptits.

The comparison of the Km and vmax values for various primers was carried out. The primers were either completely complementary to the poly(A)-template or contained noncomplementary bases in different positions from the 3'-end. An increase of the Km and vmax values for primers containing noncomplementary bases was shown. The affinity of the AMV-revertase complex with poly(A)-template to d(pT)10 was shown to be higher by a factor of 93, 325, 338, 425, 95 and 15 than to d(pT)9(pC), d[(pT)2pC]3pT, d(pT)8pCpT, d(pT)7pC(pT)2, d(pT)4pC(pT)5 and d(pC)3(pT)7, respectively. The vmax values for the above primers were 1.2-1.5-fold higher than for d(pT)10. The decrease of the affinity of noncorrect primers to the enzyme was supposed to serve as a mechanism for mistakes correction when noncomplementary to the template mononucleotide units were added to the primer. More effective discrimination between right and wrong primers takes place if the noncomplementary base is in the second or third position from the 3'-end. The mistake correction is performed by dissociation of a wrong primer from the complexes with the enzyme and template. The data obtained for AMV-revertase are in accord with results for pro- and eukaryotic DNA polymerases and are in favour of a similar mechanism of mistake correction by all enzymes in the case of short primers.

[RNA-dependent DNA-polymerase from avian myeloblastosis virus: effectiveness of interaction with oligothymidylate primers of various length]. / RNK-zavisimaia DNK-polimeraza iz virusa mieloblastoza ptits: éffektivnost' vzaimodeistviia s oligotimidilatami-praimerami razlichnoi dliny.

Optimal conditions for the reaction of polymerization catalyzed by RNA-dependent DNA-polymerase from AMV on poly(A)- and poly(dA)-templates with d(pT)n-primers were established. Optimal concentrations of the components and pH of the reaction mixtures were found out to differ significantly. dTTP was shown to be both a nucleotide substrate and a minimal primer of the polymerization. The Km values for d(pT)2-primer (Km = 0.11 mM and 0.54 for poly(A) and poly(dA)-templates, respectively) and longer oligothymidylates were estimated. The lengthening of d(pT)n (n = 2-10) by one mononucleotide unit led to a 3-fold and 2-fold decrease of Km value for poly(A) and poly(dA), respectively. Further lengthening of the primer (n = 10-25) did not affect Km for the primers. The maximal rates of polymerization did not depend on primer length. The activation reaction (Ea = 12 kcal/mol) of polymerization on poly(A) was considerably lower than that on poly(dA) (Ea = 50 kcal/mol). In both cases a highly processive polymerization was observed. It was suggested that the synthesis had been more effective on poly(A)-template due to a more effective formation of the complex enzyme primer template.

[The effect of bases non-complementary to the template on the efficacy of primer interaction with the Klenow fragment of DNA polymerase I from Escherichia coli]. / Vliianie nekomplementarnykh matritse osnovanii na éffektivnost' vzaimodeistviia praimerov s fragmentom Klenova DNK-polimerazy I iz Escherichia coli.

The comparison of the Km and Vmax values for the primers was carried out. The primers were either completely complementary to the template or contained non-complementary bases at different positions with respect to the 3'-end. The addition of NaF, selectively inhibiting 3'----5'-exonuclease activity of the enzyme, was shown to result in the increase of Vmax values by 10% and 30% for complementary and partially complementary primers, respectively, Km values of the latters being unchanged. Km values for d[(pT)10pC] is about 146-fold greater than that for d[(pT)11]. Km values for d[(pT)7pC(pT)2] (20 microM) and d[[(pT)2pC]3pT] (20 microM); d[(pT)4pC(pT)5] (5.0 microM); d[(pC)(pT)7] (1.3 microM) and d[(pT)2pC(pT)7] (1.2 microM) are comparable with those for d[(pT)2] (22 microM), d[(pT)5] (4.1 microM) and d[(pT)7] (1.2 microM), respectively, but not with the decathymidylate d[(pT)10] (0.2 microM). We suggest that it is not the length of the primers but the number of bases in the fragment beginning with the first nucleotide from the 3'-end and ending in the non-complementary base, that determines the efficiency of interaction of the primers containing non-complementary bases with the enzyme. The addition of one link to d(pT)n (n less than or equal to 10) resulted in a 1.8-fold increase in the affinity. When 11 less than n less than 25 the affinity is decreased so that d(pT)22-23 have minimal affinity to the enzyme. The primers containing more than 50 units were found to have about the same affinity (calculated on base concentration) as d(pT)10-11.