[A method of isolation and properties of DNA-dependent DNA-polymerase epsilon from human placenta]. / Metod vydeleniia i svoistva DNK-zavisimoi DNK-polimerazy epsilon iz plztsenty cheloveka.

DNA polymerase epsilon was purified to near homogeneity from human placenta. The enzyme has one subunit (170 kDa, sedimentation coefficient 8.2S), intrinsic 3'-5'-exonuclease activity, it is independent on PCNA and high processivity on poly(dA)-oligo(dT) template-primer without PCNA. It was shown, that the enzyme incorporates 3'-amino-2',3'-dideoxythymidine 5'-triphosphate in DNA, after that synthesis is stopped. Simultaneously DNA polymerase alpha was purified.

Formation of phosphonester bonds catalyzed by DNA polymerase.

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III.

[Formation of phosphonoester bonds, catalyzed by DNA polymerases]. / Obrazovanie fosfonoéfirnykh sviazei, kataliziruemoe DNK-polimerazami.

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma-diphosphate (II) were synthesised. Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E. coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective. Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues. Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I. The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E. coli.

[RNA and DNA synthesis catalyzed by a DNA-polymerase alpha complex with primase from silkworm cells on single-stranded DNA]. / Sintez RNK i DNK, kataliziruemyi kompleksom DNK-polimerazy alpha s praimazoi iz kletok shelkopriada na odnonitevoi DNK.

Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation.

[Fluorescent analogs of nucleoside-5'-phosphates for the study of nucleic acids by nonradioactive methods]. / Fluorestsentnye analogi nukleozid-5'-trifosfatov dlia izucheniia nukleinovykh kislot neradioaktivnymi metodami.

The synthesis of 2'-deoxyuridine 5'-triphosphate analogues with fluorescent residues of fluorescein and rhodamine nature at C5 of the uracil base was performed. Reverse transcriptase of avian myeloblastosis virus, DNA polymerase beta of rat liver, terminal deoxynucleotidyl transferase of calf thymus and E. coli DNA polymerase I, Klenow fragment, were shown to be capable to incorporate a nucleotide residue with fluorescent label into 3'-terminus of oligonucleotide. These fluorescent labeled oligonucleotides were used as primers for synthesis of (-)-chain of M13mp10 phage. Fluorescently labeling template-primer complexes were used for DNA sequencing.

[Conformation limited nucleoside-5'-phosphates as termination substrates for DNA-polymerases]. / Konformatsionno ogranichennye nukleozid-5'-trifosfaty kak terminatornye substraty DNK-polimeraz.

We have investigated the ability of some nucleoside 5'-triphosphate analogues to terminate the DNA synthesis catalyzed by calf thymus DNA polymerase alpha and terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E. coli DNA polymerase I (Klenow's fragment) and AMV reverse transcriptase. It has been shown that lyxoanhydronucleoside 5'-triphosphates terminate DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotydil transferase. 2',3'-O-Isopropylidenecytidine 5'-triphosphate inhibits the DNA synthesis catalyzed by reverse transcriptase and DNA polymerase beta and its moiety was incorporated in the place of dTMP residue. Riboanhydroadenosine 5'-triphosphate reveals the properties of an effective termination substrate for all the DNA polymerases studied. This is the first attempt to investigate nucleotide analogues with the restricted conformation of the carbohydrate moiety as termination substrates for several prokaryotic and eukaryotic DNA polymerases.

[Analogs of pyrophosphate in a pyrophosphorolysis reaction catalyzed by DNA polymerases]. / Analogi pirofosfata v reaktsii pirofosforoliza, kataliziruemoi DNK-polimerazami.

The reaction of pyrophosphorolysis catalyzed by Escherichia coli DNA polymerase I Klenov fragment, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and AMV reverse transcriptase was studied. Some pyrophosphate (PPi) analogs were taken as low molecular weight substrates. It was shown that only imidodiphosphonic acid acted as the PPi substrate analog for the reactions catalyzed by DNA polymerases I and alpha, both imidodiphosphonic acid and methylenediphosphonic acid were active in the case of DNA polymerase beta and reverse transcriptase. Other analogs tested were neither nucleotide residue acceptors, nor inhibitors of the pyrophosphorolysis reaction with PPi. The abilities of some PPi analogs to inhibit the DNA elongation catalyzed by reverse transcriptase were investigated. The principles of specificity of low molecular substrates recognition by DNA polymerases and some problems concerning the mechanisms of DNA synthesis inhibition by PPi analogues are discussed.

[Selective inhibition of 3'-5'-exonuclease activity of DNA-polymerase I from Escherichia coli by a fluoride ion]. / Selektivnoe ingibirovanie ftorid-ionom 3'-5'-ékzonukleaznoi aktivnosti DNK-polimerazy I Escherichia coli.

The effect of NaF on the enzymatic activities of the large fragment of E. coli DNA polymerase I (Klenow enzyme-KE) with different DNA-substrates was studied. It was shown that fluoride ion at concentrations of 5-10 mM efficiently inhibits the 3'----5' exonuclease activity of KE but does not affect the polymerase activity of the enzyme. Selective inhibition of the 3'----5' exonuclease activity of KE is Mg-dependent and is observed with double- or single-stranded DNAs. In reaction with the 14-mer oligonucleotide annealed with single-stranded phage M13 DNA the enzyme was found not only to perform the exonucleolytic hydrolysis of the primers but to catalyse also a limited elongation of some primers, adding a few nucleotide residues in the absence of exogenous dNTP. The primer elongation is inhibited by inorganic pyrophosphatase and is stimulated by micromolar concentrations of exogenous pyrophosphate thus suggesting a possible role of PPi contamination in dNTP generation via pyrophosphorolysis. Traces of precursors in DNA preparations obtained by generally employed methods may serve as another source of nucleotides for the primer elongation.

[Analogs of deoxyribonucleoside-5'-triphosphates modified in the base and sugar moieties: substrate properties in DNA biosynthesis in cell-free systems]. / Analogi dezoksiribonukleozid-5'-trifosfatov, modifitsirovannye po osnovaniiu i sakharnomu ostatku: substratnye svoistva pri biosinteze DNK v beskletochnykh sistemakh.

Incorporation of 2'-deoxyribonucleotide 5'-triphosphate derivatives, chemically modified both in the base and at 3'-position, into DNA by four different DNA polymerases was investigated. It is shown that 3'-azido- and 3'-amino-2',3'-dideoxy-(E)-5-(2-bromovinyl)-uridine 5'-triphosphates effectively terminate DNA synthesis catalyzed by E. coli DNA polymerase I, rat liver DNA polymerase beta, and AMV reverse transcriptase. Calf thymus DNA polymerase alpha incorporates only the 3'-amino derivative. DNA polymerases I and beta catalyse DNA synthesis in the presence of beta-D-(2'-deoxyribofuranosyl)-1-benzimidazol 5'-triphosphate, inserting the corresponding monophosphate in place of -dGTP, whereas 3'-substituted analogues of this compound were inactive in the reactions.

[Incorporation, into the DNA, chain of a fluorescent derivative of 2-deoxyuridylic acid during catalysis of synthesis by DNA-polymerase A]. / Vkliuchenie v tsep' DNK fluorestsentnogo proizvodnogo 2'-dezoksiuridilovoi kisloty pri katalize sinteza DNK-polimerazoi A.

A 2'-deoxyuridine 5'-triphosphate analogue with a dansyl (5-dimethylaminonaphtalene 1-sulphonyl) residue in the 5-position of uracyl has been synthesised. This compound substitutes dTTP in the DNA synthesis catalyzed by Klenow's fragment of E. coli DNA polymerase I on the M13mp10 phage DNA as template with synthetic 14-member primer. When the synthesis is terminated by four termination substrates, structure of the synthesised DNA chain can be read. It demonstrates in principle possibility of determination of DNA sequence by means of fluorescence.

[Substrate inhibitors of DNA biosynthesis]. / Substratnye ingibitory biosinteza DNK.

We present an account of the data on the inhibition analysis of DNA biosynthesis by substrate analogs with DNA polymerases of different origin. An attempt has been made to substantiate the factors that underline the specificity of inhibitors with respect to DNA synthesis that is catalyzed by various DNA polymerases.

Characterization of 3'----5' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.

[DNA polymerase beta from the rat liver. Isolation, properties and inhibitory analysis of a homogeneous preparation]. / DNK-polimeraza beta iz pecheni krysy. Poluchenie, svoistva i ingibitornyi analiz gomogennogo preparata.

A simple and reproducible purification procedure of homogeneous DNA polymerase beta from rat liver is developed, including sedimentation and saline extraction of rat liver chromatin, chromatography of the extract on DEAE-cellulose, phosphocellulose, Gel Blue A, and DNA sepharose. The purified enzyme isolated with the 8.4% yield proved to be a homogeneous protein with m.w. 38-40 kDa, specific activity 31 units/g, pI 8.6-8.9. Incorporation of [3H]TTP into activated DNA catalysed by DNA polymerase beta was strongly inhibited by dNTP (3'NH2), ddTTP, dNTP (3'F) and slightly inhibited by aCTP and aNTP (3'NH2).

2',3'-Dideoxy-3' aminonucleoside 5'-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.