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V M Krutiakov

Publications and source records attributed to V M Krutiakov.

At least 19 recordsLinked to original sources
PubMed1989

[Analysis of proteins in normal and regenerating rat liver using two-dimensional electrophoresis].

The major proteins of homogenate, cytosol, nuclei and nuclear membrane extract from normal and regenerating rat liver were studied by two-dimensional electrophoresis with a view of detecting proteins involved in DNA replication regulation. Essential quantitative differences in three out of 200 polypeptides separated as spots and dyed with Coomassie R-250 on two-dimensional maps were revealed. The content of the p38 nuclear protein (Mr congruent to 38 kD, pI congruent to 4) increases 6-8-fold in the S-phase. The level of another nuclear protein, p50 (Mr congruent to 50 kD, pI congruent to 6.5) decreases 2-3-fold. The cytoplasmic protein p35 (Mr congruent to 35 kD, pI congruent to 8) also decreases 2-3-fold. Moreover, the p40 protein (Mr congruent to 40 kD, pI congruent to 6) whose content in the nuclei sharply rises up to 20 times after sham operation was revealed.

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PubMed1986

[Cloning of mRNA nucleotide sequences amplified during DNA replication in the regenerating liver].

A library of double-stranded cDNA has been constructed using the mRNA of regenerating rat liver 20 hr after partial hepatectomy. The differential screening of the library with the regenerating liver specific and the resting-liver-specific single-stranded cDNA probes has identified 11 cDNA clones which sequences are preferentially expressed in regenerating rat liver. The RNA dot blot hybridization has shown that levels of RNA complementary to these clones are 3 to 8-fold higher in dividing cells as compared with resting cells.

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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.

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PubMed1982

[Non-specific acid nucleoside triphosphatase from cytosol and chromatin of rat liver: partial purification and general properties].

The nucleoside triphosphatase (EC 3.6.1.15) was isolated from rat liver cytosol and purified 600-fold. The enzyme hydrolyzes all NTPs and dNTPs, splits NDPs and dNDPs at a low rate and does not destroy NMPs and dNMPs. The phosphatase consists of a single subunit with molecular weight of 65 000. The chromatin fraction of the enzyme is fully bound to the membrane, whereas the cytosol fraction contains 15-30% of the membrane-bound enzyme. Both free and membrane-bound phosphatases possess identical functional properties. The enzymatic activity has a pH-optimum of 4.0--4.5, is increased in the presence of Me2+ and is unaffected by ouabain, Triton X-100, N-ethylmaleimide, NaF or DNA, but is inhibited by NaCl, Pi and PPi. The value of Km is equal to 20 microM for TTP splitting. Since the NTP pool is essentially changed throughout the cell cycle, it is suggested that the nucleoside triphosphatase can participate in the nucleotide pool regulation.

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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.

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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.

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PubMedDate not supplied

[Antimutagenic role of autonomous 3'-->5'-exonucleases].

Our own and literary data about antimutagenic role of autonomous 3'-->5'-exonucleases (AE) are analyzed. AE are not bound covalently to DNA polymerases but often involved in replicative complexes. Intracellular overproduction of AE in bacteria is accompanied with the sharp suppression of mutagenesis, whereas the inactivation of AE in bacteria and higher fungi results in the increase of mutation rates by 2-3 orders of magnitude. The addition of AE in biologically meaningful concentrations to DNA polymerases elevates substantially the accuracy of their work in vitro. In these cases, the reverse mutation rates were measured in the DNA from phage (X174 amber 3, whereas the direct mutation rates--in the DNA from phage M13mp2, both being used as primer-templates for DNA synthesis and then transfected into spheroplasts of Escherichia coli. The accuracy of action of nuclease-free DNA polymerases alpha and beta are shown to raise in the presence of AE by 2-3 orders, the accuracy of moderately processive DNA polymerase I--by 2 orders, the accuracy of highly processive DNA polymerase delta--by 5-10 times, though the latter 2 polymerases display and their own 3'-->5'-exonucleolytic activity. AE, involved in the multienzyme DNA polymerase complexes, augment the accuracy of complexes action by 5-10 times. The model of "external" corrective role of AE in DNA biosynthesis is proposed. Study of 30 objects from all 3 kingdoms of live beings (from archae- and eubacteria to mammalia including human) has shown that AE account, as minimum, from 30 to 90% of the total cellular 3'-->5'-exonucleolytic activity. So AE increase essentially the intracellular ratio of values of 3'-->5'-exonuclease to DNA polymerase activities in the very various representatives from a phylogenetic tree that results always in the augmentation of the accuracy of DNA biosynthesis.

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