Effect of bases noncomplementary to the template on the effectiveness of primer interaction with DNA-polymerase alpha from the human placenta

The comparison of the Km and Vmax values for the various primers was carried out. The primers were either completely complementary to the template or contained the non-complementary bases in different positions from the 3'-end. The number of the bases from the 3'-end to the noncomplementary nucleotide but not the primers length was supposed to determine the efficiency of the interaction of the primers containing noncomplementary bases with the enzyme. The Km values for d[(pC) (pT)7] (1.2 microM), d[(pC)3(pT)7] (2.5 microM, d[(pT)2pC(pT)7] (1.4 microM)d[(pT)4pC(pT)5(4.3 microM); d[(pT)7pC(pT)2] (11 microM) are comparable with the Km values for d(pT)7 (1.4 microM); d(pT)5 (4.2 microM) and d(pT)3 (15 mkM), respectively, but not for the decathymidilate d[(Tp)9T] (0.23 microM). The complementary interaction between the first nucleotide from the 3'-end of the primer and the template appear to play the particular role in the interaction of the enzyme with the primer. The Km values for d[(pT)10pC] and d[(pA)9pC] (with the corresponding templates) are 38 and 6 times the ones for d[(Tp)10T] and d(pA)10. However, the Km values for d[(pA)9p(rib)] (0.56 microM) which contains the deoxyribozylurea residue at the 3'-end is practically equal to the Km for d(pA)9 (0.56 microM). The Vmax values for d[(pT)10pC] and d[(pA)9pC] are 1.7 and 2.3 times the values for d[(Tp)10T] and d(pA)10, respectively. The primer affinity decreases, just as its conversion rate increases when the noncomplementary base in the primer is transferred from the 5'-to 3'-end; that results in the rate of primers elongation decrease in total.     

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

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.     

Comparison of initiating abilities of primers of different length in polymerization reactions catalyzed by DNA polymerases from thermoacidophilic archaebacteria

Optimal conditions for polymerization reaction catalyzed on poly(dA) and poly(dT) templates by DNA polymerases from thermoacidophilic archaebacteria--DNA polymerase A from Sulfolobus acidocaldarius and DNA polymerase B from Thermoplasma acidophilum--have been established. Values of Km and Vmax (60 degrees C) for a set of primers d(pA)n and d(pT)n have been estimated. Minimal primers for both enzymes are dNMP. Lengthening of primers by each mononucleotide increases their affinity about 2.16-fold. Linear dependence of log Km and of log vmax on the number of mononucleotide links in primers (n) has breaking point at n = 10. The value of Vmax is about 20% of that for decanucleotide. The affinity of the primer d(pA)9p(rib*) with a deoxyribosylurea residue at the 3'-end does not differ essentially from that of d(pA)9. Substitution of the 3'-terminal nucleotide of a complementary primer for a noncomplementary nucleotide, e.g., substitution of 3'-terminal A for C in d(pA)10 in the reaction catalyzed on poly(dT), decreases the affinity of a primer by one order of magnitude.     

The algorithm of estimation of the Km values for primers of various structure and length in the polymerization reaction catalyzed by Klenow fragment of DNA polymerase I from E. coli

DNA synthesis at primers d(pT)n, d(pA)n, d(pC)n, and d(pG)n in the presence of corresponding complementary templates and at hetero-oligoprimers complementary to M13 phage DNA was investigated. The values of both -log Km and log Vmax increased linearly if homo-oligoprimers contained less than 10 nucleotides. The lengthening of d(pT)n and d(pA)n primers by one mononucleotide unit (n = 1-10) resulted in the 1.82-fold decrease of the Km values. The incremental decreases of Km for d(pC)n and d(pG)n were equal to about 2.46. The enhancement of the homo- and hetero-oligonucleotide primers' affinity to the enzyme due to one Watson-Crick hydrogen bond between complementary template and primer is about 1.35 times. This allows to calculate the Km values for primers of various structure and length up to 10 units. The objective laws of the Km and Vmax values changes for primers containing more than 10 nucleotides were analyzed.     

Dependence of 3'-5-exonuclease activity of a fragment of Klenow DNA polymerase I from Escherichia coli on the length and structure of the cleaved oligonucleotide

The Km and vmax values for oligothymidylates d(pT)2-16 in reaction of 3'-5'-exonuclease hydrolysis catalyzed by Klenow fragment were measured in the absence and presence of poly(dA) template without the poly(dA), the Km values for oligonucleotides are slightly dependent on their length. The rate of oligothymidylates hydrolysis increases with their length and for d(pT)16 it is about 190-times higher than for d(pT)2. The addition on poly(dA) does not lead to an essential change of the Km values for d(pT)2-16, but raises the rate of d(pT)2-7 hydrolysis 2-17-fold and at the same time lowers the efficiency of d(pT)8-16 hydrolysis. The Km values for d(pC)10, d(pA)19 and d(pT)10 are nearly the same. However the velocity of d(pC)10 hydrolysis is approximately 1,2 and 7,8-times higher than for d(pA)10 and d(pC)10, respectively d(pC)10, d(pA)10 and d(pT)10 under conditions of interaction with the template-binding site raise the rate of hydrolysis of d(pT)2 combined with the exonuclease center, with various efficiency. Under similar conditions, d(pT)8, d(pT)10 and d(pT)16 as templates activated hydrolysis of d(pT)2. The dependence of the Klenow fragment exonuclease activity both on the length and structure of the template and on the length of the hydrolyzed oligonucleotide was suggested.     

RNA-dependent DNA-polymerase from avian myeloblastosis virus: effectiveness of interaction with oligothymidylate primers of various length]

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.     

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.     

Comparison of the effectiveness of the interaction of ribo- and deoxyriboprimers with DNA-polymerase from the human placenta

The Km and Vmax values for primers d(pA)n, d(pT)n, r(pA)n, r(pU)n where n = 1-16, were compared. The Km values for minimal primers dTMP, dAMP, rUMP, rAMP were found to be 48, 71, 602 and 602 microM, respectively. The Vmax value for any NMP made up approximately 7% of that for (pN)10. The lengthening of any primer per one mononucleotide unit for n from 1 to 10 resulted in the decrease of the Km value 1.8-fold and the increase of the Vmax value 1.35-fold. The ratios of the Km values for primers r(pA)n-d(pA)n and r(pU)n-d(pT)n were 7.5 and 12.5, respectively, for any n. The Km value for [d[pT)8]r(pU) primer was the same as for r(pU)9, but not for d(pT)9. Decanucleotide [d(Tp)9]ddT interacted with the polymerase competitively to the template, but not to the primer. The primer's 3'-OH group was supposed to form the hydrogen bond with the enzyme. The absence of 3'-hydroxygroup in [d(Tp)9]ddT resulted in its inability to compete effectively with the primer. The difference of the affinity of ribo- and deoxyriboprimers is due, apparently, to the existence of the different conformation of the furanose rings in the ribose and deoxyribose.     

Structure-function analysis of mononucleotides and short oligonucleotides in the priming of enzymatic DNA synthesis

The reversed-phase chromatography technique was employed in the measurement of DNA synthesis at the primers d(pT)n, r(pU)n, d(pA)n, and r(pA)n (n = 1-16) in the presence of template poly(dA) or poly(dT). DNA synthesis was catalyzed by Escherichia coli DNA polymerase I Klenow fragment, Physarum polycephalum DNA polymerase beta-like, P. polycephalum DNA polymerase alpha, and human placenta DNA polymerase alpha. Values of Km and Vmax were measured as functions of the primer chain lengths. It was found that all mononucleotides and small oligonucleotides served as primers of DNA synthesis. Values of the logarithm of both Km and Vmax increased linearly until primers had attained a chain length of 9-12 nucleotides, where a break was observed. The incremental as well as the absolute values of Km were interpreted in terms of free binding energies. These together with other data indicate that the 3'-ultimate nucleotide of the primer contributes a decisive amount of free energy of binding to DNA polymerase both from the nucleoside and from the phosphate moiety. The incremental increase is due to a complementary interaction between bases of primer and template buried in the binding cleft of the polymerase. It is also the ultimate nucleotide that determines whether the ribonucleotide or the deoxyribonucleotide is an efficient primer. It is of interest that the major results seem preserved for all four DNA polymerases. An energetic model for the binding of the template-primer was proposed and compared with available crystallographic data.     

Utilization of mismatched initiator termini by avian myeloblastosis virus DNA polymerase.

DNA synthesis by avian myeloblastosis virus was studied using poly(C) as template and modified oligo(dG) as primer. The addition of one noncomplementary base to the 3'-end of the primer has no important effect on synthesis. The mispaired base is incorporated into the product and the apparent Km (for primer) and the V of the reaction remain unchanged. This confirms the absence of a 3' leads to 5'-exodeoxynuclease activity using a template that is transcribed faithfully rather than one that can undergo a slippage reaction.     

The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization.

Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family. Kinetic analysis of inhibition of pol alpha with mixtures of complementary and noncomplementary template:primers indicated that both nucleotides induced the formation of a polymerase: inhibitor:primer-template complex. Primer extension experiments using the guanine form as the model analog indicated that pol alpha cannot utilize these nucleotides to extend primer termini. In contrast, pol T4 polymerized BuPdGTP, indicating that resistance to polymerization is not a common feature of the inhibitor mechanism among the broad membership of the alpha polymerase family.     

DNA-polymerase alpha from human placenta. Effectiveness of interaction between oligothymidylates of different lengths and the template-binding site

Modification of human placenta DNA polymerase alpha by (pT)2pC[Pt2 + (NH3)2OH].(pT)7 was investigated. The linear time dependence of the enzyme activity logarithm suggested a pseudo-first order for modification. Kd value of enzyme-affinity reagent complex (0.5 microM) was estimated. The enzyme inactivation by the affinity reagent and protection from inactivation in the presence of oligonucleotides of varying length were used for determining Kd values of the enzyme-ligand complexes. Oligonucleotide d(pT)2pC(pT)7 (Kd 0.15 microM), d(Tp)9T (Kd 0.15 microM) and [d(Tp)9]ddT (Kd 0.15 microM) protected the enzyme from inactivation with equal efficiency. The protective action of oligothymidylates d(Tp)nT (where n changes from 3 to 14) strongly depended on the chain length, the Kd values diminishing from 5.3 to 0.0091 microM in the geometrical progression. The addition of one link to the oligothymidylate chain resulted in 1.71-fold increase in the oligonucleotide affinity for the enzyme specific site. Such a change corresponds to Gibbs energy change of about 0.32 kcal/mole. It is supposed that the monomer units of pentadecathymidylate (at least beginning with the third one) in d(Tp)14T-enzyme complex form neither hydrogen bonds nor electrostatic linkages with the enzyme. Kd values of oligonucleotides as templates are shown to reflect quite well the true affinity of template for the enzyme. This affinity increases in the presence of a primer. However, the ratio of the affinity for different oligonucleotides does not change in the presence or absence of a complementary primer.     

E. coli DNA polymerase. A study of the mechanism of primer binding using oligothymidylate analogs with ethylated internucleotide phosphate groups

The following individual diastereomers of oligothymidylate ethyl esters (the alkyl phosphodiester group is asymmetric with R or S configuration) have been prepared: d[(Tr)8Tp'(Et)T] (I), d[(Tp)8Tp'(Et)T] (II), d[(Tp)8Tp'(Et)TpT] (III), d[(Tp)8Tp' X (Et)TpT] (IV). A totally esterified analogue d[[(Tp(Et)7]T] (V) was obtained as a diastereomeric mixture. All oligothymidylate derivatives revealed substrate activity as primers of DNA polymerase with poly(dA) as a template. The values of the maximal reaction rates were equal to 14; 2,6; 68; 24 and 0,1% for oligothymidylates (I)-(V) with respect to Vm value (100%) for (Tp)9T. Km values of oligothymidylates (I)-(V), 2,7; 2,5; 0,51; 7,2 microM, were obtained in relation to Km for d[(Tp)9T] (0,4 microM). Diastereomers (I) and (II) were not destroyed by Klenow fragment of DNA polymerase I which has only 3'----5' exonuclease activity. However, these derivatives were hydrolyzed by complete DNA polymerase I due to its 5'----3' exonuclease activity, the reaction rate being 3-10 times lower than in case of d[(Tp)9T]. The data suggest an essential contribution to the primer binding from the positive enzyme group interaction with the 3'-end negatively charged phosphate group of oligonucleotide, together with the primer complementary interaction with the template. At least two phosphodiester groups of the oligonucleotide primer are essential for the reaction of polymerization following the correct binding.     

Eukaryotic and prokaryotic DNA-polymerase. II. The role of internucleotide phosphate groups of a template in its binding with the enzyme

The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated. To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC[Pt2+(NH3)2OH](pT)7 on the concentration of these ligands as competitive inhibitors were determined. Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM). Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14). The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme. However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times. The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions. The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place. The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole. Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole). The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site. Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit. We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site. Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E. coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.     

The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data.

Pt(2+)-containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E. coli and DNA polymerase alpha from human placenta. The values of Kd and Gibb's energy (delta G degree) for the complexes of oligodeoxyribonucleotides and their derivatives with the template sites of these enzymes were determined from the effects protecting the enzyme from inactivation by Pt(2+)-containing oligonucleotides. Kd and delta G degree values of the complexes made by DNA polymerases and orthophosphate, triethylphosphate, d(pC)n, d(pT)n, d(pG)n, d(pA)n (where n = 1-25), heterooligonucleotides of various length and structure, and oligothymidylates with partially and completely ethylated internucleotide phosphates were evaluated. The obtained data enabled us to suggest 19-20 mononucleotide units of the template to interact with the protein. Only one template internucleotide phosphate forms a Me(2+)-dependent electrostatic contact (delta G = -1.1...-1.7 kcal/mol) and a hydrogen bond (delta G = -4.4...-4.9 kcal/mol) with the enzyme. It is likely that the mononucleoside units of the template form hydrophobic contacts with the enzymes. The efficiency of such interaction changes with the hydrophobicity of the bases: C less than T less than G approximately A. For both homo- and heterooligonucleotides the contributions of nucleoside units to the affinity of the templates to the enzymes is due to the complementary interactions with the primers. A hypothetical model for the template-primer interaction with DNA polymerases is suggested.     

DNA polymerase alpha cofactors C1C2 function as primer recognition proteins

Most, if not all, of the DNA polymerase alpha activity in monkey and human cells was complexed with at least two proteins, C1 and C2, that together stimulated the activity of this enzyme from 180- to 1800-fold on low concentrations of denatured DNA, parvovirus DNA, M13, and phi X174 DNA or RNA-primed DNA templates, and poly(dT):oligo(dA) or oligo(rA). These primer-template combinations, which have from 200 to 5000 bases of template/primer, were then 7- to 50-fold more effective as substrates than DNase I-activated DNA. C1C2 specifically stimulated alpha polymerase, and only from the same cell type. Alpha X C1C2-polymerase reconstituted from purified alpha polymerase and the C1C2 cofactor complex behaved the same as native alpha X C1C2-polymerase and C1C2 had no effect on the sensitivity of alpha polymerase to aphidicolin, dideoxythymidine triphosphate, and N-ethylmaleimide. In the presence of substrates with a high ratio of single-stranded DNA template to either DNA or RNA primar, C1C2 increased the rate of DNA synthesis by decreasing the Km for the DNA substrate, decreasing the Km for the primer itself, increasing the use of shorter primers, and stimulating incorporation of the first deoxyribonucleotide. In contrast, C1C2 had no effect on the Km values for deoxyribonucleotide substrates (which were about 150-fold higher than for DNA replication in isolated nuclei), the ability of specific DNA sequences to arrest alpha polymerase, or the processivity of alpha polymerase. Accordingly, C1C2 function as primer recognition proteins. However, C1C2 did not reduce the comparatively high Km values or stimulate DNA synthesis by alpha polymerase on lambda DNA ends and DNase I-activated DNA, substrates with 12 and about 30-70 bases of template/primer, respectively. DNA restriction fragments with 1 to 4 bases of template/primer were substrates for neither alpha nor alpha X C1C2-polymerase. Therefore, we propose that C1C2 enhances the ability of alpha polymerase to initiate DNA synthesis by eliminating nonproductive binding of the enzyme to single-stranded DNA, allowing it to slide along the template until it recognizes a primer.     

Interactions of calf thymus DNA polymerase alpha with primer/templates.

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.