Biomedical subjects

O I Lavrik

Publications and source records attributed to O I Lavrik.

At least 19 recordsLinked to original sources

PubMed1992

Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.

FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.

Affinity Labels↗

PubMed1992

Human immunodeficiency virus type 1 reverse transcriptase. Affinity labeling of the primer binding site.

Affinity modification of the primer site of HIV1-RT was performed with an oligonucleotide derivative containing a photoreactive azido group at the 5' end of d(pT)10. The affinity of HIV1-RT for d(pT)10 and for its derivative was first estimated by measuring the Michaelis constants of these two oligonucleotides acting as primers in the retrotranscription of poly(rA). The enzyme was then inactivated under UV-irradiation at 303-365 nm in the presence of ArN3-d(U*T9); the dependence of the rate of inactivation on primer concentration was found to be consistent with the Km value. Last, selectivity of affinity modification was demonstrated through elongation of the covalently bound primer and selective protection of inactivation by d(pT)10 or tRNA(Lys).

Affinity Labels↗

PubMed1992

Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA(Phe).

Phenylalanyl-tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNA(Phe). It is shown that the 'hyperaminoacylated' tRNA(Phe) is the bis-2',3'-O-phenylalanyl-tRNA(Phe), and its formation is typical for the thermophilic enzyme but does not occur for E. coli phenylalanyl-tRNA synthetase under the same conditions.

Autoradiography↗

PubMed1992

Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion. In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted. The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein. The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide. The pheST genes are presumably cotranscribed and translationally coupled. A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes. The genes pheS and pheST were expressed in E.coli.

Amino Acid Sequence↗

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↗

PubMed1992

A comparative study of the relationship between thermostability and function of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus.

The relationship between thermostability and functional activities of phenylalanyl-tRNA synthetases (EC 6.1.1.20) from E. coli and Thermus thermophilus has been studied. In the case of the E. coli enzyme, the activity decreased after the 43 degrees C treatment, both in the [32P]PPi-ATP exchange reaction and the overall aminoacylation reaction, due to thermo-inactivation of the phenylalanyl-tRNA synthetase, whereas tRNA(Phe) preserved its native structure. In the Th. thermophilus system, the enzyme showed extreme thermostability (up to 90 degrees C), and the reduction in the tRNA aminoacylation rate after the 78 degrees C treatment was ascribed to denaturation of the tRNA(Phe). Since the enzyme did not lose the [32P]PPi-ATP exchange activity up to 85 degrees C, the observed lower thermo-resistance of the tRNA is evidence that the native structure of ribonucleic acids should be one of the most difficult to stabilize at high temperatures.

Enzyme Stability↗

PubMed1992

Recognition nucleotides for human phenylalanyl-tRNA synthetase.

The specificity of the interaction between tRNAPhe and phenylalanyl-tRNA synthetase isolated from human placenta was investigated. Using yeast tRNAPhe transcripts with different point mutations it was shown that all the five recognition points for the yeast phenylalanyl-tRNA synthetase (G20, G34, A35, A36 and A73) are also important for the reaction catalyzed by the human enzyme. A set of mutations in nucleotides involved in tertiary interactions of tRNAPhe revealed that mutations which maintained the proper folding of the molecule had almost no influence on the efficiency of aminoacylation. The most striking difference between the yeast and human phenylalanyl-tRNA synthetases involved a mutation in the lower two base pairs of the anticodon stem. This mutation did not affect aminoacylation with the yeast enzyme, but greatly reduced activity with human phenylalanyl-tRNA synthetase.

Anticodon↗

PubMed1991

Comparative study of subunits of phenylalanyl-tRNA synthetase from Escherichia coli and Thermus thermophilus.

FPLC separation of alpha- and beta-subunits of phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8 has been carried out in the presence of urea. Native alpha-subunits of both enzymes were primarily alpha 2-dimers and tended to aggregate. Most E. coli enzyme beta-subunits were monomeric and only a small fraction was represented by beta 2-dimers. All thermophilic beta-subunits were beta 2-dimers. It was shown that monomers and all forms of homologous subunits had no catalytic activity in tRNA(Phe) aminoacylation. For the enzymes and their subunits, titration curves were obtained and isoelectric points were determined. The comparison of the relative surface charges indicated similarity of the surfaces of entire enzymes and the corresponding beta-subunits. Alpha-subunits displayed a distinctly different pH dependence of the surface charge. A spatial model of the oligomeric structure and a putative mechanism for its formation are discussed.

Chromatography↗

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

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.

Animals↗

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↗

PubMed1991

Oligonucleotides and their derivatives as tools for investigations of protein-nucleic acid interactions in template biocatalysis.

On the basis of quantitative characteristics (Kd, Km, Gibbs energy values) for the interaction of oligonucleotides with template and primer site of eucaryotic and procaryotic DNA polymerases, a general model of template-primer interaction with these enzymes was suggested. The interactions of AMV- and HIV- reverse transcriptases with various 5'-derived oligonucleotides and with human DNA polymerase alpha and Klenow fragment are compared. The results obtained suggest a method to improve selectively the affinity of an oligonucleotide primer to RNA template with AMV- and HIV-reverse transcriptases.

Avian Myeloblastosis Virus↗

PubMed1991

[Chemical modification of lysine residues in tyrosyl-tRNA-synthetase from cattle liver using pyridoxal-5'-phosphate].

Chemical modification of lysine residues of eukaryotic tyrosyl-tRNA synthetase was studied. It was shown that only four out of 22 lysine residues per enzyme dimer could be modified with pyridoxal-5'-phosphate. This modification led to the inactivation of tRNATyr aminoacylation by more than 90% but did not practically affect the rate of ATP-[32P]pyrophosphate exchange. Low molecular weight substrates (ATP, ATP-tyrosine) weakly protected the enzyme from inactivation, whereas tRNATyr afforded a much more effective protection. It was supposed that lysine residues of tyrosyl-tRNA synthetase can be involved in the interaction with tRNATyr.

Animals↗

PubMed1991

Diadenosine oligophosphates: peculiarities of synthesis by phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8.

Temperature and other factors affecting synthesis of bis(5'-adenosyl) tetraphosphate (Ap4A) and bis(5'-adenosyl)triphosphate (Ap3A) catalyzed by phenylalanyl-tRNA synthetases (PheRSs) from Escherichia coli MRE-600 and Thermus thermophilus HB8 have been investigated. Those two synthetases exhibited different temperature-dependent rates of the Ap4A and Ap3A synthesis. However, with respect to the effects of such effectors of the Ap4A synthesis as Zn2+, Mg2+, tRNA and Ap4A phosphonate analogues, as well as some inhibitors of aminoacyl-tRNA synthetase, those two enzymes were apparently undistinguishable.

Dinucleoside Phosphates↗

PubMed1990

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.

Avian Myeloblastosis Virus↗

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

[A comparative study of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus by the tritium topography method].

A comparative study of thermostability and amino acid composition of phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. In the thermophilic protein the proline, leucine, phenylalanine, arginine content was considerably increased, whereas that of asparagine, isoleucine, serine, threonine and lysine was decreased as compared to the mesophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of the both enzymes. In the E. coli enzyme the threonine residues were also easy to access, while on the surface of the thermophilic enzyme arginine residues were more abundant. A quantitative assay of the surface compositions revealed the increased exposure of (Leu + Ile) residues in the thermophilic protein as well as of the charged asparagine and arginine residues. A possible relationship of the observed effects to thermostability is discussed.

Enzyme Stability↗

PubMed1979

[Modification of the alpha-subunit of phenylalanyl-tRNA synthetase from E. coli MRE-600 with N-chlorambucilyl-phenylalanyl-tRNA].

L-Phenylalanyl-tRNA synthetase from E. coli MRE-600 (EC 6.1.1.20) was alkylated with N-chlorambucilyl-[14C] phenylalanyl-tRNA. After removal of the affinity reagent tRNA moiety bp alkaline hydrolysis of the ester bond between the N-chlorambucilyl-phenylalanyl residue and the 3'-end of tRNA, The enzyme was dissociated into subunits in the presence of SDS. Separation of the subunits was performed by SDS electrophoresis. The bulk of the radioactivity of the N-chlorambucilyl-[14C] phenylalanyl residue was found at the position of the alpha-subunit of the enzyme. The results obtained are consistent with a specific binding of the phenylalanyl-tRNA analog to the alpha-subunit of the enzyme followed by covalent binding of the N-chlorambucilyl-phenylalanyl moiety to the protein.

Affinity Labels↗