Biomedical subjects

V N Podust

Publications and source records attributed to V N Podust.

At least 37 records · Page 2Linked to original sources

PubMed1993

Comparison of interactions of 5'-derivatives of deoxyoctathymidylate with human DNA polymerize alpha and HIV reverse transcriptase.

Km and Vmax values for d(pT8) and its derivatives containing various 5'-end groups were estimated in the reaction of DNA polymerization alpha catalyzed by DNA polymerase alpha and HIV-RT. The effect of 5'-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT8 is 200-fold higher than that of d(pT8). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase alpha the influence of modifiers on primer affinity is much weaker. The effect of 5'-end residues on the Vmax values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.

DNA↗

PubMed1993

[Study of the interaction of DNA primase from calf thymus and human placenta with oligonucleotides matrices of various length and structure].

Human placenta DNA-primase as a component of the DNA-polymerase alpha-primase multienzyme complex was examined with a view of establishing the dependence of Km values in the reaction of oligoriboadenylate synthesis from ATP on the length of a poly(dT) template. The pKm values increased linearly up to ten monomeric units of the oligo(dT)n template. These data favour oligo(dT)10 as an optimal template covered by the active site of this enzyme. The DNA-primase catalyzed processively the synthesis at each polymerization cycle of a unique length primer (7-10 nucleotides) as follows from the analysis of the primer length and its distribution with time. It is suggested that the 10 mer DNA-RNA duplex of the template and the primer is a critical size for dissociation of primase and further elongation of the primer by DNA-polymerase in the presence of dNTP.

Animals↗

PubMed1992

Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.

Animals↗

PubMed1992

Eukaryotic DNA primase appears to act as oligomer in DNA-polymerase-alpha--primase complex.

Human placenta and calf thymus DNA-polymerase-alpha-primases were analyzed using native gradient-polyacrylamide-gel electrophoresis followed by overlay assays of polymerase and primase activities. The human enzyme contained three catalytically active native forms of 330, 440 and 560 kDa and the bovine enzyme five forms of 330, 440, 500, 590 and 660 kDa. Of the various DNA polymerase forms, only the largest (560 kDa for human DNA polymerase and 590 kDa and 660 kDa for bovine DNA polymerase) contained primase activity. Titration of human DNA-polymerase-alpha-primase with DNA-polymerase-free primase caused the conversion of the 440-kDa to the 560-kDa form. The data favour the idea that primase binds to DNA polymerase alpha as an oligomer of 3 primases/polymerase core. In addition, the ability of primase to utilize oligoriboadenylates containing (prA)n or pp(prA)n was investigated. The primase elongated pp(prA)2-7 up to nanoadenylates or decaadenylates, but did not add 9 or 10 mononucleotides to a preexistent primer. In contrast to pp(prA)n less than 10, (prA)n less than 10 were rather poor primers for the primase. Both pp(prA)8,9 and (prA)n greater than 10 were elongated by primase, producing characteristic multimeric oligonucleotides. The possible connection of the structure of the DNA-polymerase-alpha-primase complex with the catalytical properties of primase is discussed.

Animals↗

PubMed1991

Eukaryotic DNA primase. Abortive synthesis of oligoadenylates.

Calf thymus DNA polymerase alpha-primase, human placenta DNA polymerase alpha-primase and human placenta DNA primase synthesized oligoriboadenylates of a preferred length of 2-10 nucleotides and multimeric oligoribonucleotides of a modal length of about 10 monomers on a poly(dT) template. The dimer and trimer were the prevalent products of the polymerization reaction. However, only the oligonucleotides from heptamers to decamers were elongated efficiently by DNA polymerase alpha.

Adenine Nucleotides↗

PubMed1991

Human placenta DNA primase: purification of enzyme and analysis of RNA primer synthesis.

The immunoaffinity purification of human placenta DNA primase devoid of DNA polymerase alpha activity is described. Primase consists of 52 and 59 kDa polypeptides. They form a single protein of 330 kDa under native conditions. The polypeptide structure of primase is believed to be (52 + 59)3. Primase synthesizes the oligoribonucleotides 2-9 monomers long and multimeric oligoribonucleotides of a modal length of about 10 monomers. The following model of RNA primer synthesis is proposed: 1) primase, being in free state or in complex with Pol alpha, forms a protein trimer or another structure that includes several primases; 2) primase synthesizes de novo only the oligonucleotides 2-10 monomers in length; 3) the newly synthesized oligonucleotides dissociate in solution or translocate to either Pol alpha or a neighbouring primase unit to be further elongated with the next 7-10 mononucleotides.

Chromatography, Affinity↗

PubMed1990

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.

Base Sequence↗

PubMed1990

Inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate allows estimation of the primers affinity.

Template-primer dependent inactivation of human DNA polymerase alpha and Klenow fragment of E. coli DNA polymerase I by adenosine 2',3'-riboepoxide 5'-triphosphate was used for quantitative analysis of the Kd values for oligonucleotide primers of different length. The Kd values are smaller by a factor of 2.5 than the Km values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase alpha. The Kd and Km values are nearly the same for Klenow fragment. Such approach to the determination of Km/Kd ratio can likely be used for detailed quantitative analysis of DNA polymerases.

Adenosine Triphosphate↗

PubMed1990

[Template-primer-dependent inactivation of DNA polymerase alpha from human placenta by 2',3'-epoxyadenosine-5'-triphosphate].

Modification of the human placenta DNA polymerase alpha by 2',3'-epoxyadenosine 5'-triphosphate (eATP) was investigated. The latter binds to the protein both in absence and in presence of template-primer complex. However for inactivation of the enzyme, reagent-complementary template, primer and Me2(+)-ions are required. The inactivation is apparently due to the affinity modification of dNTP-binding site by eATP; covalent binding of the reagent off the enzyme's active site without affecting the DNA polymerase activity is also suggested. The enzyme inactivation by eATP and its protection from inactivation in the presence of dATP were used to determine Kd values of complexes of the enzyme with eATP (90 microM) and dATP (1 microM), the latter value being 13-times lower than Km for dATP (13 microM) in the polymerisation reaction. Using the dependence of the DNA polymerase inactivation by eATP on the primer concentration, Kd for enzyme-primer complexes were estimated. The Kd value for d(pA)10 (0.33 microM) was close to Km value (0.43 microM) for this primer. eATP was concluded to be a useful reagent for estimating the efficiency of the complex formation of different ligands with dNTP- and primer-binding sites of DNA polymerase.

Adenosine Triphosphate↗

PubMed1989

The efficiency of interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with the active centre of E. coli DNA polymerase I Klenow fragment.

The interaction of deoxyribonucleoside-5'-mono-, di- and triphosphates with E. coli DNA polymerase I Klenow fragments was examined. Dissociation constants of the enzyme complex with nucleotides were determined from the data on the enzyme inactivation by adenosine 2',3'-riboepoxide 5'-triphosphate. The role of nucleotide bases, phosphate groups and sugar moieties in the complex formation of nucleotides with the enzyme was elucidated. The necessity of complementary interaction of nucleotides with templates for template-controlled 'adjusting' of complementary dNTP to its reactive state was found. The crucial role of the interaction of dNTP gamma-phosphate with the enzyme in this process is discussed.

Binding Sites↗

PubMed1989

DNA polymerase alpha-DNA primase from human placenta. Immunoaffinity purification and preliminary characterization.

Highly purified DNA polymerase alpha-DNA primase from normal human tissue (human placenta) has been prepared by immunoaffinity purification on immobilized anti-human DNA polymerase alpha monoclonal antibody SJK 287-38. According to data from SDS electrophoresis this preparation consists of subunits of 180, 160, 145, 140 kDa (a cluster of DNA-polymerizing subunits), 73 kDa (function unknown) and 59, 52 kDa (corresponding to primase). Three active enzyme forms of 270, 460 and 575 kDa have been revealed using native electrophoresis followed by detection of DNA polymerase activity.

Chromatography, Affinity↗

PubMed1987

The efficiency of dNTP complex formation with human placenta DNA polymerase alpha as demonstrated by affinity modification.

The interaction of deoxyribonucleoside 5'-mono-, di- and triphosphates with human placenta DNA polymerase alpha was examined. Dissociation constants of enzyme complex formation with dNMP, dNDP and dNTP were determined from the data on enzyme affinity modification by imidazolide of dTMP. The basic role of the primary template-primer interaction with the enzyme in dNTP complex formation is shown. The template-dependent nucleotide interaction does not occur in the case of dNMP and dNDP in comparison with dNTP. The significant contribution of the gamma-phosphate of dNTP in this process is demonstrated.

Binding, Competitive↗

PubMed1987

Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase alpha.

Affinity labelling of human placenta DNA polymerase alpha (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)nT where n = 1-14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+-dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.

DNA Polymerase II↗

PubMed1987

[Prokaryotic and eukaryotic DNA-polymerase. I. The role of internucleotide phosphate groups in the binding of a primer with the enzyme].

The mechanism of binding and elongation of the oligothymidylate primers in the systems of the DNA polymerase alpha from human placenta and DNA polymerase I from E. coli with the poly(dA) as a template was investigated. Both dTMP and dTTP were shown to be the minimal primers of DNA polymerase alpha, the affinity and V increasing 1.8- and 1.4-fold respectively upon lengthening the primer by each unit from dTMP to d(Tp)9T. Further elongation is accompanied by 1.3-fold affinity enhancement and a decrease in V. For the E. coli enzyme, a similar dependence of affinity of primer d(Tp)4T-d(Tp)14T was observed with the inflexion point corresponding to d(Tp)8T. The individual diastereomers of oligothymidylate ethyl esters (with p' and p'' corresponding to enantiomeric configuration) such as d[Tp'(Et)Tp]3Tp'(Et)T, d[Tp''(Et)Tp]3Tp''(Et)T, d(Tp)8Tp'(Et)T, d(Tp)8Tp''(Et)T, d(Tp)8Tp'(Et)TpT, d(Tp)8 X X Tp''(Et)TpT and completely esterified analogues d[Tp(Et)]7T, d[Tp(Et)]14T were shown to initiate the poly (dA)-dependent polymerization catalyzed by both enzymes. A sum of the obtained results provided the basis for a number of conjectures on the mode of primer and template binding to the enzyme, possible role of their preformed complex, as well as electrostatic interactions and hydrogen bonding.

DNA Polymerase I↗

PubMed1987

[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.

DNA Polymerase I↗

PubMed1986

[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.

Affinity Labels↗

PubMedDate not supplied

[Purification and characterization of DNA-dependent DNA-polymerase alpha from human placenta].

A preparation of human placenta DNA polymerase with specific activity 6000 unit/mg was obtained. The protocol of the enzyme purification includes the crude extract preparation, the subsequent chromatographies on phosphocellulose, red sepharose, DEAE sepharose and hydroxylapatite. The isolated DNA polymerase belongs to alpha-type according to the large molecular mass (greater than 150 kDa), high sensitivity to N-ethylmaleimide, the profound inhibition of DNA polymerization activity by 200 mM KCl and the ability to catalyze DNA synthesis, using the deoxyribonucleic template and ribonucleic primer. The DNA polymerase preparations contain a few forms with Stokes radii 50-60 A and sedimentation coefficients 7.3-9.0 S as found from data of gel-filtration and ultracentrifugation in glycerol density gradient, accordingly. The existence of four various forms of DNA polymerase activity: 150, 170, 220, 480 kDa were revealed by native electrophoresis. The four steps of purification result in DNA polymerase preparation that was shown by electrophoresis to contain 15-20% of protein possessing the polymerase activity. However the preparation obtained seems to be a "chromatographically pure substance", according to following ion-exchange and affinity chromatographies. The other proteins without polymerase activity are suggested to be the components of the replicative complex of human placenta cells.

Chromatography, Affinity↗

PubMedDate not supplied

[Effectiveness of complex-formation of nucleotides with human DNA polymerase alpha from data of enzyme modification by reactive nucleotide analogs].

The modification of the human placenta DNA polymerase alpha by the imidazolides of dNMP was investigated. The modification was shown to occur only in the simultaneous presence of the template and the primer. This process, however, doesn't depend on the complementary interaction of the nucleotide base with the template. The Kd values of the complexes between the different nucleotides and DNA polymerase alpha were estimated. The affinity of Im-dTMP was determined from the dependence of the Kapp of the enzyme inactivation rate on the reagent concentration. The Kd values for dNMP, dNDP, dNTP were estimated using the protective effect of these nucleotides under the enzyme modification by Im-dTMP. The comparison of the interaction efficiency between the polymerase and dNMP, dNDP, dNTP (complementary or non-complementary to the template) allow to conclude that the nucleotide discrimination occurs on the dNTP level, i. e. dNMP and dNDP upon forming the complex with the enzyme, don't interact complementarily with the template. The additional contacts between the enzyme and the nucleotide terminal phosphate were supposed to form only for the complementary dNTP. The studies allowed to put forward a hypothetical model of the template complementary dNTP binding to the polymerases. The role of the hydrophobic interaction of the nucleotides with the enzyme as well as the possible influence of the nucleotide gamma-phosphate group on the template--dNTP complement formation. The Watson-Crick bound formation of the nucleotide with the template was supposed to be followed by the additional conformational rearrangement of the nucleotide triphosphate chain. The latter process leads to the formation of additional contacts between the enzyme and the nucleotide gamma-phosphate.

Catalysis↗