[Isolation of total RNA from baker's yeast].

A simple method of production of total RNA from baker's yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100 degrees C for 40-60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.

Hydrolytic approach for production of deoxyribonucleoside- and ribonucleoside-5 -monophosphates and enzymatic synthesis of their polyphosphates.

A new, simple, and ingenious method for enzymatic synthesis of deoxy- and ribonucleoside-5 -triphosphates (dNTP and NTP, respectively) has been developed. The method includes the following stages: hydrolysis of DNA with DNase and immobilized S1-nuclease, phosphorylation of the resulting deoxy- and ribonucleoside-5 -monophosphates (dNMP and NMP, respectively) with nucleotidyl kinase from Escherichia coli, and purification by chromatography of the synthesized dNTP and NTP. dNMP was phosphorylated using an ATP-regenerating system based on acetokinase from E. coli and lithium acetylphosphate.

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.

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.

[Affinity modification of DNA polymerase I from Escherichia coli and its Klenow fragment with nucleotide imidazolides]. / Affinnaia modifikatsiia DNK-polimerazy I iz Escherichia coli i ee fragmenta Klenova imidazolidami nukleotidov.

Affinity modification of E. coli DNA polymerase I and its Klenow fragment by imidazolides of dNMP (Im-dNMP) and dNTP was studied. DNA polymerase activity of DNA polymerase I was reduced by both Im-dNMP and Im-dNTP. However Im-dNTP does not inactivate of the Klenow fragment. The level of covalent labelling of both enzymes by radioactive Im-dNTP did not exceed 0.01 mol of reagent per mol of enzyme. But the deep inactivation of DNA polymerase I by Im-dNTP was observed. It is likely that this inactivation is due to the formation of intramolecular ether followed by phosphorylation of the carboxyl group. This assumption is strongly supported by the increase of the isoelectrical point of DNA polymerase I after its incubation with Im-dNTP in conditions of enzyme inactivation. All data permit us to suggest that the affinity modification of both enzymes by Im-dNMP and covalent labeling by Im-dNTP takes place without complementary binding of dNTP moiety with the template. However inactivation of DNA polymerase I by Im-dNTP occurs only if the dNTP-moiety is complementary to the template in the template.primer complex. It was shown that His residue was phosphorylated by Im-dNMP and Tyr or Ser residues between Met-802 and Met-848 were phosphorylated by Im-dNTP. We suppose that there are two states of DNA polymerase active site for the binding of dNTPs. One of them is independent on the template, in the other state the dNTP hydrogen bond with the template is formed.

NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment.

It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.

Interaction of dNTP, pyrophosphate and their analogs with the dNTP-binding sites of E. coli DNA polymerase I Klenow fragment and human DNA polymerase alpha.

The 3',5'-exonuclease center of the Klenow fragment of E. coli DNA polymerase I (FK) was selectively blocked by NaF. The latter was shown to forbid the binding of nucleotides and their analogs to the enzyme exonuclease center. In the presence of poly(dT).r(pA)10 template.primer complex and NaF, we observed AMP, ADP, ATP, PPi and dATP to be competitive inhibitors of the FK-catalyzed DNA polymerization. The interactions of the nucleotides with FK and human DNA polymerase alpha were compared to reveal similarity of binding to the DNA polymerizing centers. Structural components of dNTP and PPi playing key roles in forming complexes with pro- and eukaryotic DNA polymerases were identified.

Highly selective affinity labeling of the primer-binding site of E. coli DNA polymerase I.

Highly selective affinity labeling of the primer site of E. coli DNA polymerase I was performed with the 5'-reactive derivatives of oligothymidylate in the presence of poly(dA) template. Subtilysine cleavage proved that the site of affinity modification belonged to the 'Klenow' part of DNA polymerase I. If taken separately, Klenow fragment was not labeled by these oligonucleotide derivatives. The site of affinity labeling were tested in the structure of DNA polymerase I by hydroxylamine cleavage. At least two sites of labeling were revealed. The main one was localized between Gly-833 and His-928.

[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]. / Zavisimost' 3'-5'-ékzonukleaznoi aktivnosti fragmenta klenova DNK- polimerazy I iz Escherichia coli ot dliny i struktury rasshchepliaemogo oligonukleotida.

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.

[Study of the mechanism of mutagenesis directed by phosphotriester analogs of oligonucleotides. The role of repair in mutagenesis]. / Issledovanie mekhanizmov mutageneza, napravlennogo fosfotriéfirnymi analogami oligonukleotidov. Rol' reparatsii v zakreplenii mutatsii.

The mutation system has been suggested in an effort to test insertion and deletion mutants by changing the Lac-phenotype of bacterial colonies transformed by mutant DNA. This system also makes possible to determine heterozygotes and homozygotes among the mutants. The yield of mutants in shown to depend on the structure of the DNA heteroduplex region. The yield of deletion mutants is greater than that of insertion mutants. Heterozygotes prevail in mutant colonies (greater than 90%).

[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.

[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 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.

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.

DNA polymerase I (Klenow fragment): role of the structure and length of a template in enzyme recognition.

The values of Kd and Gibbs energy (delta G degrees) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo-oligonucleotides d(pC)n, d(pT)n, and d(pA)n and hetero-oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+ (NH3)2OH](pT)7 and d[(Tp2)C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357-369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+-dependent electrostatic contact and a hydrogen bond with the enzyme. The 19-20-nucleotide fragments of the template appear to interact with the protein molecule.

[Modification of tyrosine residues of the Klenow fragment of DNA-polymerase I from Escherichia coli by acetylimidazole]. / Modifikatsiia klenovskogo fragmenta DNK-polimerazy I iz Escherichia coli atsetilimidazolom po ostatkam tirozina.

The modification of tyrosine residues of DNA polymerase I Klenow fragment from E. coli by acetylimidazole has been investigated. This reagent was shown to inactivate both polymerization and 3',5'-exonuclease activities but with different velocity. The poly(dT)-template and r(pA)10-primer each added separately to the enzyme have no notable influence on the rate of enzyme inactivation. Simultaneous presence of both template and primer increases the rate of inactivation. In the presence of poly(dT).r(pA) 10 there is not effect of dCTP and dTTP (noncomplementary to the template) on the rate of inactivation of polymerization activity. However, dATP complementary to the template, provides a complete protection. A weak protective action is detected in the presence of dADP. Orthophosphate, pyrophosphate and dAMP each taken separately increase the rate and the level of the enzyme inactivation. dAMP together with either ortho- or pyrophosphate have the same protective action as ATP. All data obtained allow to suggest the functional significance for polymerization activity of tyrosine located in the dNTP binding site of DNA polymerase I.

[Behavior of the large fragment of DNA polymerase I (the Klenow fragment) during fractionation of a cell-free extract of E. coli MRE-600]. / Povedenie bol'shogo fragmenta DNK-polimerazy I (fragmenta Klenova) pri fraktsionirovanii beskletochnogo ékstrakta E. coli MRE-600.

Distribution of the DNA polymerase I large fragment (Klenow fragment) was studied during fractionation of the E. coli MRE-600 cell-free extract with polyethylenimine. On the basis of the results obtained a simple procedure is proposed that enables the Klenow fragment to be obtained as a coproduct of DNA polymerase I, RNA polymerase, polynucleotide phosphorylase, nucleotide kinases with acetokinase and nucleoside deoxy-ribosyltransferase in the framework of a combined technological scheme.