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I V Shevelev

Publications and source records attributed to I V Shevelev.

18 recordsLinked to original sources
PubMed2000

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver.

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.

Animals↗
PubMed1999

[Constitutive inhibition of DNA degradation due to the enzyme RecBCD in the radiation-resistant Escherichia coli K-12 mutant Gam(r)444].

Exonucleolytic degradation of [3]H-labeled DNA was examined in partially purified fractions of lysates obtained from nonirradiated RecBCD enzyme-containing cells of Escherichia coli and in the radiation-resistant mutant Gamr444. The degradative activity was shown to be lowered in these cells to the same extent as in the recBC mutant. The efficiency of plating of the mutant phage T4 2-, DNA of which can be degraded by exonuclease V, was 400-fold higher on the strain Gamr444 than on the wild-type strain AB1157. This value was shown to be only twice as low as that on the recB mutant or on the strain AB1157 carrying plasmid pGam26 with a radiation-resistance allele gam26 cloned from mutant Gamr444. The data obtained confirmed the hypothesis that the Gamr444 mutant contains a constitutive inhibitor of exonucleolytic activity of the RecBCD enzyme in nonirradiated cells. This inhibitor was shown to be encoded by the gam26 allele that had previously been mapped at 56.8 min of the E. coli chromosome. A possible mechanism of the involvement of this inhibitor in enhanced radiation resistance of the mutant Gamr444 is considered.

Alleles↗
PubMed1999

[Hot start of the polymerase chain reaction using DNA helicase].

A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.

DNA Helicases↗
PubMed1996

'External' proofreading of DNA replication errors and mammalian autonomous 3'-->5'exonucleases.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. The base substitutions misinserted by these polymerases could be eliminated with two kinds of an 'external' proofreading carried out (1) by the 3'-->5' exonuclease function intrinsic to DNA polymerases delta and epsilon or/and (2) by the autonomous 3'-->5' exonucleases non-associated covalently with DNA polymerases. DNA polymerases delta and epsilon can be separated from autonomous 3'-->5' exonucleases by means of sedimentation. Ultracentrifugation of the nuclear extracts and cytosols from normal and regenerating rat liver as well as from total embryos has shown the bulk of the cellular 3'-->5' exonucleolytic activity is due to autonomous nucleases. Moreover, the level of such a specific activity correlates with the replicative status of the organs from adult animals: spleen > regenerating liver > normal liver > cardiac muscle > brain, maximum difference being an order of magnitude. In addition, autonomous exonucleases were shown to be the constituents of the multienzyme forms of DNA polymerases alpha and beta. Hence, autonomous 3'-->5' exonucleases seem to be the principal participants in an 'external' proofreading.

Animals↗
PubMed1993

Proof-reading 3'-->5' exonucleases isolated from rat liver nuclei.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-5) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A-T)] template, respectively, 10-fold and 2-fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polymerase activities being similar. Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.

Animals↗
PubMed1982

[Effect of the antitumor antibiotic bleomycin on DNA polymerase activity].

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.

Animals↗
PubMedDate not supplied

[The correcting role of autonomous 3'-->5' exonucleases in mammalian multienzyme DNA polymerase complexes].

A study was made of the correcting role of autonomous 3'-->5' exonucleases (AE) contained in multienzyme DNA polymerase complexes of rat hepatocytes or calf thymocytes. DNA was synthesized on phage psi X174 amber3 or M13mp2 primer-templates, and used to transfect Escherichia coli spheroplasts. Frequencies were estimated for direct and reverse mutations resulting from mistakes made in the course of in vitro DNA synthesis. The mistake rate of the hepatocytic complex was estimated at 3 x 10(-6) with equimolar dNTP, and increased tenfold when proteins accounting for 70% of the total 3'-->5' exonuclease activity of the complex were removed. The fidelity of DNA synthesis was completely restored in the presence of exogenous AE (epsilon subunit of E. coli DNA polymerase III). Nuclear (Pol delta n) and cytosolic (Pol delta c) forms of DNA polymerase delta were isolated from calf thymocytes. The former was shown to contain an AE (TREX2) absent from the latter. As compared with Pol delta c, Pol delta n had a 20-fold higher exo/pol ratio and allowed 4-5 times higher fidelity of DNA synthesis. The mistake rate of DNA polymerase complexes changed when dNTP were used in nonequimolar amounts.

Animals↗
PubMedDate not supplied

[3'-->5'-exonucleases of the rat liver and the correction of replication errors].

Mammalian nuclear DNA polymerases alpha and beta are lack of the proofreading 3'-->5' exonucleolytic activity. 40 and 50 kDa 3'-->5' exonucleases were isolated from rat liver. The exonucleases were shown to excise mismatched nucleotides from poly[d(A--T)] template 10 and 2 fold faster than matched ones. The addition of either exonuclease to DNA polymerase alpha from rat liver or calf thymus 5-10 times increased the accuracy of reproduction of primed DNA from bacteriophage phi X174 amber 3, values of exonuclease and DNA polymerase activities being approximately equal. The exonuclease activity surpasses the DNA polymerase one by an order of magnitude in chromatin and nuclear membrane. These data, taken together, are indicative of potent proofreading into hepatocytes.

Animals↗
PubMedDate not supplied

[Molecular reasons for the regulation of the enzymatic activity of bovine pancreatic deoxyribonuclease I as studied by dynamic light scattering and fluorescent spectroscopy].

The effect of catalytic bivalent and inhibitory monovalent cations on the interactions of bovine pancreatic deoxyribonuclease I with the circle single-stranded DNA of M13 phase was studied. It was found that monovalent cations affect the site of binding to DNA and the active center of the enzyme; in their presence, a break of the formation of the enzyme-substrate complex occurs. The results provide evidence that conformational changes of the protein molecule in all cases are more substantial that it could be expected from X-ray data published earlier.

Animals↗
PubMedDate not supplied

[Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver].

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.

Animals↗
PubMedDate not supplied

[Isolation of protein phosphokinase from a complex form of DNA polymerase alpha from rat liver].

Protein kinase activity was revealed in complex forms of rat liver DNA polymerase alpha containing 3'-5'-exonuclease, primase, helicase, DNA ligase. Protein kinase (mol. mass about 200 kDa) has been partially purified from a specimen of high molecular mass DNA polymerase alpha of nuclear membrane of regenerating liver. The protein kinase activity of the complex form of DNA polymerase alpha was maximal in the cytosol in normal rat liver cells and in the nuclear membrane in dividing cells (40 h after partial hepatectomy). The main phosphokinase properties of this enzyme were determined.

Animals↗
PubMedDate not supplied

[Contribution of 3'---5'-exonuclease from rat liver nuclei in precision of DNA synthesis, catalyzed by mammalian DNA polymerase alpha].

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of editing 3'-->5'exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5'exonucleases with molecular masses of 40 and 50 kDa have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from a poly [d(A-T)] template respectively 10- and 2-fold faster than the matched ones. Upon addition of any of these exonucleases to DNA polymerase alpha from rat liver or calf thymus, the fidelity of in vitro reproduction of primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, the levels of exonuclease and polymerase activities being approximately the same. The extrapolation of replication fidelity to cellular activities of the exonucleases and alpha-polymerase suggests that exonuclease proofreading augments the accuracy of DNA synthesis at least by three orders of magnitude.

Animals↗