Biomedical subjects

E S Gromova

Publications and source records attributed to E S Gromova.

At least 19 recordsLinked to original sources

PubMed2004

Homology modeling of the CG-specific DNA methyltransferase SssI and its complexes with DNA and AdoHcy.

Prokaryotic DNA methyltransferase M.SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed "FRankenstein's monster" approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.

2-Aminopurine↗

PubMed2000

Parallel self-associated structures formed by T,C-rich sequences at acidic pH.

Oligonucleotides of nonregular heteropyrimidine sequences incorporating or not incorporating purine residues 5'-d(ACTCCCTTCTCCTCTCTA), 5'-d(ACTCCCTGGTCCTCTCTA), 5'-d(TCTCTCCTGGTCCCTCC), and 5'-d(TCTCTCCTCTTCCCTCC) can form self-associated parallel-stranded (ps) structures at pH 4-5.5. The ps structures were identified by studying at neutral and acidic pH UV melting transitions, FTIR spectra, and fluorescence of pyrene-labeled oligonucleotides as well as by chemical joining of 5'-phosphorylated oligonucleotides. A gel electrophoresis run for oligonucleotides 5'-d(TCTCTCCTCTTCCCTCC) and 5'-d(ACTCCCTTCTCCTCTCTA) has shown the formation of homoduplexes at low DNA strand concentrations. Ps structures are held by C-C(+) base pairs and have N- and S-types of sugar puckering as detected by FTIR spectroscopy in the millimolar concentration range. Guanine inserts as well as thymine and purine inserts into an oligomeric cytosine sequence make the formation of the tetraplex i-motif unfavorable. MvaI restriction endonuclease, which recognizes the CCT/AGG sequence in DNA, does not cleave parallel pseudosubstrates.

Acids↗

PubMed2000

DNA duplexes containing altered sugar residues as probes of EcoRII and MvaI endonuclease interactions with sugar-phosphate backbone.

Oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (dTx) in place of dC and dT residues in the EcoRII and MvaI recognition site CC(A/T)GG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2'-deoxyxylosyl moieties of dCx and dTx, 3'-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2'-deoxyxylosyl moiety into a dC x dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA x dT pair the effect of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dCx modifications and their combination with dTx did not enhance the destabilization effect. Hydrolysis of dCx-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dTx residue was cleaved by the enzyme, but kcat/K(M) was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.

Carbohydrates↗

PubMed2000

A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis.

Site-directed mutagenesis of the ecoRII gene has been used to search for the active site of the EcoRII restriction endonuclease. Plasmids with point mutations in ecoRII gene resulting in substitutions of amino acid residues in the Asp110-Glu112 region of the EcoRII endonuclease (Asp110 --> Lys, Asn, Thr, Val, or Ile; Pro111 --> Arg, His, Ala, or Leu; Glu112 --> Lys, Gln, or Asp) have been constructed. When expressed in E. coli, all these plasmids displayed EcoRII endonuclease activity. We also constructed a plasmid containing a mutant ecoRII gene with deletion of the sequence coding the Gln109-Pro111 region of the protein. This mutant protein had no EcoRII endonuclease activity. The data suggest that Asp110, Pro111, and Glu112 residues do not participate in the formation of the EcoRII active site. However, this region seems to be relevant for the formation of the tertiary structure of the EcoRII endonuclease.

Amino Acid Sequence↗

PubMed1998

The Ecl18kI restriction-modification system: cloning, expression, properties of the purified enzymes.

Ecl18kI is a type II restriction-modification system isolated from Enterobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransferase (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have been cloned and expressed in Escherichia coli. These enzymes recognize the 5'.../CCNGG...3' sequence in DNA; M.Ecl18kI methylates the C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as shown by the arrow. The restriction endonuclease and the methyltransferase were purified from E. coli B834 [p18Ap1] cells to near homogeneity. The restriction endonuclease is present in the solution as a tetramer, while the methyltransferase is a monomer. The interactions of M.Ecl18kI and R.Ecl18kI with 1,2-dideoxy-D-ribofuranose containing DNA duplexes were investigated. The target base flipping-out mechanism is applicable in the case of M.Ecl18kI. Correct cleavage of the abasic substrates by R.Ecl18kI is accompanied by non-canonical hydrolysis of the modified strand.

Base Sequence↗

PubMed1998

EcoRII endonuclease has two identical DNA-binding sites and cleaves one of two co-ordinated recognition sites in one catalytic event.

EcoRII is a typical restriction enzyme that cleaves DNA using a two-site mechanism. EcoRII endonuclease is unable to cleave DNA which contains a small number of EcoRII recognition sites but the enzyme activity can be stimulated in the presence of DNA with a high frequency of EcoRII sites. To investigate the mechanism of activation, the kinetics of stimulated EcoRII cleavage has been studied. A 14 bp substrate activated the cleavage of the 71 bp substrate, containing one EcoRII recognition site (trans-activation) by a competitive mechanism: the activator increased substrate binding but not catalysis. The activation increased if the substrate concentration decreased and if the activator had a lower affinity for the enzyme than the substrate. The introduction of the second recognition site into the 71 bp duplex also enabled cleavage of this substrate (cis-activation). Pyrophosphate bonds were incorporated into one of two recognition sites to switch off the cleavage of the phosphodiester bonds. Analysis of cleavage products of these modified substrates showed that EcoRII cuts one of two coordinated recognition sites in one catalytic event.

Base Sequence↗

PubMed1997

DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction- modification enzymes.

To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regioselective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-beta-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the Eco RII and Mva I R-M enzymes that contained a galactose or periodate-oxidized galactose residue as single substituents either in the center of the Eco RII (Mva I) recognition site or in the flanking nucleotide sequence. The dependence of binding, cleavage and methylation of these substrate analogs on the modified sugar location in the duplex was determined. Cross-linking of the reagents to the enzymes under different conditions was examined. M. Eco RII covalent attachment to periodate-oxidized substrate analogs proceeded in a specific way and to a large extent depended on the location of the reactive dialdehyde group in the substrate. The yield of covalent attachment to a DNA duplex with a dialdehyde group in the flanking sequence with Eco RII or Mva I methylases was 9-20% and did not exceed 4% for R. Eco RII.

Aldehydes↗

PubMed1996

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs.

Specific and non-specific interactions of SsoII restriction endonuclease (R.SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R.SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R.SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg2+ ions.

Base Sequence↗

PubMed1996

[Kinetic modeling of the mechanism of allosteric interactions of restriction endonuclease EcoRII with two DNA segments].

The effect of correlations between kinetic parameters of two inducible substrates on allosteric activation of EcoRII endonuclease hydrolysis of one substrate was studied. The pairs of DNA duplexes were constructed that were the substrates of EcoRII restriction endonuclease or their analogs and had different kinetic constants of interaction with the enzyme; the effects of their concentrations on mutual hydrolysis induction were studied. A kinetic mechanism is suggested considering the allosteric effects of two DNA recognition sites on dimeric molecule of EcoRII. Mathematic modeling was used to analyze the kinetic mechanism and evaluate optimal characteristics of the inductor. Thus, activation increases when (i) substrate concentration decreases, (ii) enzyme binding of two inductor or substrate molecules decreases, (iii) binding of one substrate molecule increases versus binding of one inductor molecule, and (iv) kcat of the enzyme-substrate complex including on substrate and one inductor increases.

Allosteric Regulation↗

PubMed1995

The interaction of DNA duplexes containing 2-aminopurine with restriction endonucleases EcoRII and SsoII.

Oligonucleotides containing 2-aminopurine (2-AP) in place of G or A in the recognition site of EcoRII (CCT/AGG) or SsoII (CCNGG) restriction endonucleases have been synthesized in order to investigate the specific interaction of DNA with these enzymes. Physicochemical properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-like form. 2-Aminopurine base paired with cytidine, however, essentially influences the helix structure. The presence of a 2-AP-C mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a biphasic melting behaviour. SsoII restriction endonuclease recognizes and cleaves the modified substrate with a 2-AP-T mismatch in the centre of the recognition site, but it does not cleave the duplexes containing 2-aminopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-aminopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-aminopurine in place of adenine in the presence of the canonical substrate.

2-Aminopurine↗

PubMed1995

Chemical cross-linking of MvaI and EcoRII enzymes to DNA duplexes containing monosubstituted pyrophosphate internucleotide bond.

DNA duplexes containing a monosubstituted pyrophosphate internucleotide group, instead of a phosphodiester bond, were used as cross-linking reagent for the affinity modification of the restriction endonucleases EcoRII and MvaI (R.EcoRII and R.MvaI). An active group was introduced into the enzyme's recognition site or between the recognition site and flanking sequence. The substrate properties of such DNA duplexes were determined. Cross-linking specificity was demonstrated by competition experiments with unmodified substrate, as well as by the absence of cross-linking to an active duplex lacking a recognition site. It was shown that the nucleophilicity of the buffer solution and the presence of the enzyme cofactor Mg2+ dramatically affected the cross-linking yield.

Base Sequence↗

PubMed1995

DNA duplexes containing methylated bases or non-nucleotide inserts in the recognition site are cleaved by restriction endonuclease R.EcoRII in presence of canonical substrate.

DNA duplexes containing the natural methylated bases N6-methyladenine (m6Ade), N4-methylcytosine (m4Cyt) or C5-methylcytosine (m5Cyt) in one strand of the recognition sequence are resistant to EcoRII restriction endonuclease (R.EcoRII). Hydrolysis of these modified duplexes was observed in the presence of the canonical substrate. Incorporation of m4Cyt or m5Cyt into both strands of the recognition sequence precludes such activation by a canonical substrate. R.EcoRII also fails to cleave substrate analogs in which one of the nucleosides in the recognition site is replaced by the 1,2-dideoxyribose (D) or by 1,3-propanediol (Prd) (modeling DNA with an abasic site). The hydrolysis of DNA duplexes with non-nucleotide inserts is also activated in the presence of canonical substrate. Thus, the two-substrate mechanism of EcoRII-DNA interaction allows hydrolysis of apurinic/apyrimidinic and hemimethylated DNA.

5-Methylcytosine↗

PubMed1995

Interaction of the MvaI and SsoII methyltransferases with DNAs altered at the central base pair of the recognition sequence.

The interaction of the MvaI and SsoII DNA methyltransferases (MTases; M.MVaI and M.SsoII, respectively) with a set of synthetic DNA duplexes, containing a M.MvaI and M.SsoII recognition site (CCWGG), was investigated. In these DNA duplexes dA or dT of the recognition site was replaced by nucleoside analogs with modified sugar moieties and heterocyclic bases (2'-deoxy-2'-fluorouridine (flU), 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (xT), 1-(beta-D-3'-deoxy-threo-pentofuranosyl)uracil (tU)), or by 1,3-propanediol (Prd). A new approach for monitoring methylation of each strand of DNA duplexes by MTases was developed. It allowed the determination of the influence of the modification in one DNA strand on the methylation of the other. In most cases, for both M.MvaI and M.SsoII, sugar analog-containing duplexes showed inhibition of methylation of only the modified strand. Prd-containing DNA duplexes were not substrates for M.MvaI. M.SsoII did not methylate DNA duplexes in which the dT residue was replaced by Prd.

Amino Acid Sequence↗

PubMed1995

Modified substrates as probes for studying uracil-DNA glycosylase.

In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(beta-D-2'-deoxy-threo-pentofuranosil)uracil (xU) or 1-(beta-D-3'-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2'-deoxy-2'-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100-200-fold less efficiently than the natural ones. F or Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.

Base Sequence↗

PubMed1995

Kinetic studies of MvaI DNA methyltransferase interaction with modified oligonucleotide duplexes.

We have measured steady-state kinetics of a N4-cytosine methylase, M.MvaI, using as substrates modified non-selfcomplementary tetradecanucleotide duplexes containing the CCWGG target sequence. The inner or outer localisation of the dI residue in the MvaI recognition site seems to be of little importance since the specificity constants kcat/KM are only 2 to 7 fold smaller than that of the canonical substrate. Replacement of dG residues by dI in both strands resulted in a 25 to 60-fold decrease of the specificity constant. Modifications of the phosphate backbone or opening of the sugar ring of one of the dG residues had only little influence on the action of M.MvaI. The enzyme appears to be rather tolerant to different kinds of modifications in its substrate in the mainor groove.

Base Sequence↗

PubMed1995

Use of UV spectroscopy for the study of nucleic acid cleavage by E. coli RNase H and restriction endonucleases.

A one-step spectrophotometric method for monitoring of nucleic acid cleavage by ribonuclease H from E. coli and type II restriction endonucleases has been proposed. It is based on recording of the increase in the UV absorbance at 260 nm during the course of enzymatic reaction. Duplexes stable under the reaction conditions were chosen as substrates for the enzymes being studied. In order to obtain duplex dissociation following their cleavage by the enzyme appreciate temperature conditions were selected. The spectrophotometric method may be applied for rapid testing of the nuclease activity in protein preparations as well as for precise quantitative analysis of nucleic acid degradation by enzymes. This method may be successfully employed in kinetic studies of nucleic acid-protein interactions.

Base Sequence↗

PubMed1994

Two subunits of EcoRII restriction endonuclease interact with two DNA recognition sites.

The cleavage of a 14 base pair DNA duplex containing one EcoRII recognition site by EcoRII restriction endonuclease (R.EcoRII) was studied in single turnover experiments with varying enzyme concentrations in the micromolar range. The reaction rate increased with enzyme concentration until a ratio of one dimeric R.EcoRII enzyme to two double stranded oligonucleotide molecules. Excess of R.EcoRII lead to inhibition of cleavage. Maximum cleavage was also found with pBR322 DNA containing six EcoRII recognition sites at a ratio of one dimeric enzyme to two EcoRII recognition sites of the plasmid DNA. At higher ratios inhibition was observed. These observations indicate that the active enzyme complex is formed when two subunits of the enzyme interact with two R.EcoRII recognition sites.

Base Composition↗

PubMed1994

[Affinity modification of EcoRII restriction endonuclease by a DNA-duplex containing a monosubstituted pyrophosphate internucleotide bond].

Oligonucleotide duplex with an active monosubstituted pyrophosphate bond within the recognition site of the EcoRII restriction endonuclease was cross-linked to this enzyme with a yield of 10-15%. The cross-linking specificity was proved by the absence of the cross-linking to a DNA duplex with the same modification but without the EcoRII recognition site as well as by unmodified EcoRII substrate's inhibition of the cross-linking.

Affinity Labels↗