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R O Anarbaev

Publications and source records attributed to R O Anarbaev.

6 recordsLinked to original sources
PubMed2000

Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates.

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.

Cations, Divalent↗
PubMed1998

Klenow fragment and DNA polymerase alpha-primase fromserva calf thymus in water-in-oil microemulsions.

The activity of DNA polymerase alpha-primase complex from calf thymus and Klenow fragment of E. coli DNA polymerase 1 has been studied in reverse microemulsions formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecylsulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), polyoxyethylene 20 cetyl ether (Brij 58), and Triton X-114 in decane. DNA polymerases were not active in AOT, CTAB, and SDS reverse microemulsions, but these enzymes catalyzed DNA synthesis in Brij 58 and its mixture with other surfactants. We have also found the system composed from the Triton X-114, SDS, CTAB, and Brij 58 (concentration of 128, 25, 15, and 10 mM, respectively) in hexanol-decane (1:12 v/v), in which DNA polymerases revealed maximum activity. The above system was optically transparent, fluid, and stable during a few hours with a water-surfactants molar ratio up to 160. The pH dependence of DNA polymerase activity was not significantly different in comparison with water; however, DNA polymerase was sensitive to ionic strength in microemulsions. The dependence of DNA polymerase activity on w0 was the curve with a few optima. DNA polymerases synthesized more products in water than in reverse microemulsions, and the processivity of Klenow fragment decreased. An increase of the water content resulted in an increase of DNA polymerase processivity.

Animals↗
PubMed1995

The interaction of synthetic templates with eukaryotic DNA primase.

The template interaction of calf thymus and human placenta DNA primases has been investigated. Using oligothymidylates, we showed that a template consisting of ten monomeric units was the critical size for interaction with the enzyme. The hydrophobic effect is likely to be a major factor determining template recognition by the DNA primase. The correlation between the template affinity with the enzyme and the octanol-water hydrophobic scale confirms this suggestion. In addition, the electrostatic interaction between the phosphate group of the template and side chains on the enzyme probably increases template affinity. Using methylated poly(dA), we found that the first nucleotide base of the primer should be more hydrophobic than the corresponding nucleotide base of the template. A model for the mechanism of action of DNA primase is suggested on the basis of data presented in this study and previous findings. According to this model, (a) DNA primase binds ten nucleotides of the template; (b) the synthesis of primer up to the formation of a decamer occurs processively and competes with template binding of the enzyme; (c) the conditions under which the nucleotides are incorporated into the RNA product change during the extension of the primer, and these changes are responsible for switching from primase to polymerase activity. The template specificity of DNA primase is likely to play an important role in the initiation and regulation of DNA replication.

Base Sequence↗
PubMed1995

Highly selective affinity labeling of DNA polymerase alpha-primase from human placenta by reactive analogs of ATP.

Highly selective affinity labeling of a DNA-polymerase alpha-primase complex from human placenta by o-formylphenyl esters of ATP, ADP and AMP was performed in a two-step procedure in which a substrate analog attached to the active center was elongated by radioactive ATP. If the covalent attachment is performed in the presence of poly(dT) template, the ATP esters modify selectively the delta subunit of the complex. If poly(dT) is added after the covalent binding of the reagent, both delta and gamma subunits become labeled. With the o-formylphenyl ester of AMP the delta-subunit is modified. The ADP ester modifies both the delta and gamma subunit in the presence and absence of template. It is shown that formylphenyl ester of ATP is not the substrate in the reaction of elongation catalyzed by primase. The data obtained suggest the binding site of initiating substrate to be located in the region of contact of the two subunits of primase. The role of the template in the formation of the active site is discussed.

Adenosine Triphosphate↗
PubMed1993

[Study of the interaction of DNA primase from calf thymus and human placenta with oligonucleotides matrices of various length and structure].

Human placenta DNA-primase as a component of the DNA-polymerase alpha-primase multienzyme complex was examined with a view of establishing the dependence of Km values in the reaction of oligoriboadenylate synthesis from ATP on the length of a poly(dT) template. The pKm values increased linearly up to ten monomeric units of the oligo(dT)n template. These data favour oligo(dT)10 as an optimal template covered by the active site of this enzyme. The DNA-primase catalyzed processively the synthesis at each polymerization cycle of a unique length primer (7-10 nucleotides) as follows from the analysis of the primer length and its distribution with time. It is suggested that the 10 mer DNA-RNA duplex of the template and the primer is a critical size for dissociation of primase and further elongation of the primer by DNA-polymerase in the presence of dNTP.

Animals↗