Biomedical subjects

N I Rechkunova

Publications and source records attributed to N I Rechkunova.

43 records · Page 3Linked to original sources

PubMedDate not supplied

[Comparative study of the efficacy of modifying DNA polymerase and DNA matrix by different photoactive groups at the 3'-end of the DNA primer].

The dependence of the modification efficiency of DNA polymerases and DNA template on the nature of photoactivatable group and the length of the linker that joins the group with the heterocyclic base of the primer 3'-terminal nucleotide was studied. The primers that contained the photoreactive groups at their 3'-termini were obtained using the rat DNA polymerase beta or the DNA polymerase from Thermus thermophilus in the presence of one of the dTTP analogues carrying the photoreactive group in position 5 of thymidine residue. After irradiating the reaction mixture with UV light and separating the modification products, the level of covalent binding of the [5'-32P]primer to DNA polymerases and template was determined. The primers containing 4-azido-2,5-difluoro-3-chloropyridyl group were shown to be the most effective in the modification of DNA polymerases.

Autoradiography↗

PubMedDate not supplied

[Photoreactive dTTP analogues as substrates for thermostable DNA polymerase from Thermus thermophilus B35].

Substrate properties of several dTTP analogues bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group attached at position 5 of uracil through linkers of various lengths, dTTP-NAB-x-dUTP (where x = 2, 4, 7-13 is the number of atoms in the linker), were studied. All the analogues are substrates for thermostable Thermus thermophilus B35 DNA polymerase in the elongation reaction of the 5'-32P-labeled primer-template complex. The kinetic parameters of some of the analogues were determined and compared with those of natural dTTP. It was shown that an increase in the linker length results in a higher efficiency of the analogue. The incorporation of NAB-x-dUMP residues into the 3'-primer end did not impede a further elongation of the chain in the presence of natural dNTP.

Azides↗

PubMedDate not supplied

[Interaction of human replication protein A with DNA-duplexes, containing gaps of varying sizes].

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3' end of a gap-flanking oligonucleotide with DNA polymerase beta, and an oligonucleotide containing a 5'-photoreactive group was chemically synthesized. The 5' end predominantly interacted with the large RPA subunit (p70) regardless of the gap size, whereas interactions of the 3' end with the RPA subunits depended both on the gap size and on the RPA concentration. Subunit p32 was mostly labeled in the case of a larger gap and a lower RPA concentration. The results confirmed the model of polar RPA-DNA interaction, which has been advanced earlier.

Base Sequence↗

PubMedDate not supplied

[Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap].

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.

DNA Polymerase beta↗

PubMedDate not supplied

[BsiI--a new unusual restriction endonuclease].

The restriction endonuclease BsiI from Bacillus sphaericus was isolated. The recognition sequence and cleavage point of enzyme BsiI have been determined as (sequence: see text). This restriction endonuclease is not an isoschizomer of any known restriction endonucleases and differs from other enzymes: it hydrolyses DNA into unsymmetrical recognition sequence.

Bacillus↗

PubMedDate not supplied

[Determination of the substrate specificity of Bpu101 restrictase with an unusual recognition segment].

A new enzyme Bpu10I was isolated from Bacillus pumilus. This enzyme is not an isoschizomer of any known restriction endonucleases. The search of possible recognition sequences was carried out in sequences ABCNiDEF (i = 0.6) on substrate DNA lambda CI857, T7, pBR322. The recognition sequence and cleavage sites of restriction endonuclease Bpu10I have been determined as CCTNAGC. GGANTCG

Bacillus↗

PubMedDate not supplied

[Effect of the structure of oligonucleotide substrates on kinetic parameters of interaction with BamHI restrictase].

Kinetic values of the BamHI endonuclease interaction with synthetic oligonucleotides, containing some defects, have been determined. These defects were: the absence of the one internucleotide phosphate in the GGATCC sequence; substitution of a phosphate linkage by a methylphosphonate one; 5'-protruding end of the double-stranded oligonucleotide substrate. Some modifications resulted in the increase of the initial rates of cleavage due to higher Vmax values for these substrates. Several structural defects in the oligonucleotide substrates have been shown to intensity the formation of productive complexes with the enzyme, which can be explained by the significant role of the polynucleotide chain kinks in the recognition process. Studies on oligonucleotides with different defects made it possible to reveal the phosphate groups essential for the interaction with BamHI endonuclease.

Base Sequence↗