PubMed Health⌕ Search

Biomedical subjects

S L Grokhovsky

Publications and source records attributed to S L Grokhovsky.

16 recordsLinked to original sources
PubMed2005

Sequence-specific minor groove binding ligands as potential regulators of gene expression in Xenopus laevis oocytes.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent gene activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the DNA-protein interactions following hormone activation. The strategy of artificial regulating of gene activity by sequence-specific minor groove binding ligands is very attractive. We have synthesized and studied the interaction with DNA of bis-linked netropsin derivatives in which two monomers are attached via short linkers in head-to-head and tail-to-tail manners. We have found that cis-diammine-platinum bridged bis-netropsin added to Xenopus oocytes media penetrates cellular and nuclear membrane and binds selectively to the MMTV promoter at the DNA segment that partly overlaps with the site recognized by glucocorticoid receptor. DNase I footprinting studies demonstrate that there are more stronger binding sites for cis-diammine-platinum bridged bis-netropsin on the naked MMTV DNA which are found to be inaccessible for its binding in oocytes.

Animals↗
PubMed2001

Binding of bis-linked netropsin derivatives in the parallel-stranded hairpin form to DNA.

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5'-CCTATATCC-3' in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis-diammine Pt(II)-bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5'-CCTATATCC-3' (I), 5'-CCTTAATCC-3' (II), 5'-CCTTATTCC-3' (III), 5'-CCTTTTTCC-3' (IV) and 5'-CCAATTTCC-3' (V) decreases in the order I = II > III > IV > V . The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.

Binding Sites↗
PubMed1998

DNA sequence recognition by bis-linked netropsin and distamycin derivatives.

We studied the interaction of cis-diammine Pt(II)-bridged bis-netropsin, cis-diammine Pt(II)-bridged bis-distamycin and oligomethylene-bridged bis-netropsin with synthetic DNA fragments containing pseudosymmetrical AT-rich nucleotide sequences and compared it with the interaction of the parent compounds netropsin and distamycin A. For fragments containing multiple blocks of (AIT)4 and (T/A)4 separated by zero, one, two and three GC-base pairs, DNase I footprinting and CD spectroscopy studies reveal that 5'-TTTTAAAA-3' is the strongest affinity binding site for cis-diammine Pt(II)-bridged bis-netropsin and bis-distamycin. They both bind less strongly to a DNA region containing the sequence 5'-AAAATTTT-3'. Netropsin, distamycin A and oligomethylene-bridged bis-netropsin exhibit far less sequence discrimination.

Base Sequence↗
PubMed1997

Hairpin polyamides that use parallel and antiparallel side-by-side peptide motifs in binding to DNA.

Pt-bis-netropsin is a synthetic sequence-specific DNA-binding ligand comprizing two netropsin-like fragments which are linked in a tail-to-tail manner via a cis-diammineplatinum (II) residue. The CD studies and thermodynamic characterization of the DNA-binding properties exhibited by this compound reveal that it forms two types of complexes with poly[d(AT)].poly[d(AT)] and DNA oligomers containing nucleotide sequences 5'-CC(TA)n CC-3', with n = 4, 5 and 6. The first type corresponds to the binding of Pt-bis-netropsin in the extended conformation and is characterized by the saturating ratio of one bound Pt-bis-netropsin molecule per 9 AT-base pairs. The second type of the complex corresponds to the binding of Pt-bis-netropsin to DNA in the folded hairpin form. The binding approaches saturation level when one Pt-bis-netropsin molecule is bound per four or five AT-base pairs. The hairpin form of Pt-bis-netropsin complex is built on the basis of parallel side-by-side peptide motif which is inserted in the minor DNA groove. The CD spectral profiles reflecting the binding of Pt-bis-netropsin in the hairpin form are different from those observed for binding of another bis-netropsin with the sequence Lys-Gly-Py-Py-Gly-Gly-Gly-Py-Py-Dp, where Py is a N-propylpyrrole amino acid residue and Dp is a dimethylaminopropylamino residue. The hairpin form of this bis-netropsin is formed on the basis of antiparallel side-by-side peptide motif. The CD spectra obtained for complexes of this polyamide in the hairpin form with poly[d(AT)].poly[d(AT)] exhibit positive CD band with a peak at 325 nm, whereas the CD spectral profiles for the second complex of Pt-bis-Nt with poly[d(AT)].poly[d(AT)] and short DNA oligomers have two intense positive CD bands near 290 nm and 328 nm. This reflects the fact that two bis-netropsins use different structural motifs on binding to DNA in the hairpin form.

Amino Acid Sequence↗
PubMed1996

Design of sequence-specific DNA binding ligands that use a two-stranded peptide motif for DNA sequence recognition.

The design and DNA binding activity of beta-structure-forming peptides and netropsin-peptide conjugates are reported. It is found that a pair of peptides-S,S'-bis(Lys-Gly-Val-Cys-Val-NH-NH-Dns)-bridged by an S-S bond binds at least 10 times more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT). This peptide can also discriminate between 5'-GpG-3' and 5'-GpC-3' steps in the DNA minor groove. Based on these observations, new synthetic ligands, bis-netropsins, were constructed in which two netropsin-like fragments were attached by means of short linkers to a pair of peptides-Gly-Cys-Gly- or Val-Cys-Val-bridged by S-S bonds. These compounds possess a composite binding specificity: the peptide chains recognize 5'-GpG-3' steps on DNA, whereas the netropsin-like fragments bind preferentially to runs of 4 AT base pairs. Our data indicate that combining the AT-base-pair specific properties of the netropsin-type structure with the 5'-GpG-3'-specific properties of certain oligopeptides offers a new approach to the synthesis of ligands capable of recognizing mixed sequences of AT- and GC-base pairs in the DNA minor groove. These compounds are potential models for DNA-binding domains in proteins which specifically recognize base pair sequences in the minor groove of DNA.

Aminoglycosides↗
PubMed1995

Mono-, di- and trimeric binding of a bis-netropsin to DNA.

An unusual 3:1 stoichiometry for complex formation between an elongated bis-netropsin compound and its binding site on DNA has been observed. Circular dichroism measurements distinguish two types of complexes formed between this bis-netropsin and poly[d(A-T)].poly[d(A-T)]. The first type is characterized by a 1:1 saturating ratio of bound molecules per ten base pairs. Formation of the second type results from the cooperative binding of two additional bis-netropsin molecules to the first type of complex. In contrast to these results observed for binding to the alternating polynucleotide, only the 1:1 type of complex is formed when this ligand binds to the homopolymer poly(dA).poly(dT).

Circular Dichroism↗
PubMed1991

Sequence-specific cleavage of double-stranded DNA caused by X-ray ionization of the platinum atom in the Pt-bis-netropsin--DNA complex.

An analog of the antibiotic netropsin containing two netropsin-like fragments linked covalently via a platinum atom has been synthesized. DNase I and hydroxyl radical footprinting studies have shown that this compound binds at selective sites on a DNA restriction fragment with a known nucleotide sequence. After X-ray irradiation of Pt-bis-netropsin--DNA complexes a platinum-mediated cleavage of DNA is observed at specific DNA sites. This enables one to determine the location of the synthetic ligand on the DNA with a precision of about one nucleotide. The cleavage activity seems to be related to the emission of Auger electrons from the platinum atom that cause rupture of the deoxyribose residues on the two DNA strands near the position of the platinum atom in the complex.

Autoradiography↗
PubMed1991

Design and synthesis of sequence-specific DNA-binding peptides.

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified alpha helix-turn-alpha helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha-helical, beta-sheet and random-coiled conformations with the alpha-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an alpha - beta transition in the DNA binding alpha helix-turn-alpha helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7-37 of 434 cro repressor. Evidently, in the complex, residues present in helices alpha 2 and alpha 3 of the two helix motif form a beta-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA).poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator Op1, located in the cro gene, than to the operator OR3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide alpha 2-helices. Removal of two residues from the N-terminus of helix alpha 2 in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.

Amino Acid Sequence↗
PubMed1979

DNA-binding antibiotics. X-ray structure of the distamycin A analog.

The crystal structure of the antibiotic distamycin A analog containing two pyrrol carboxamide fragments has been determined. The space group of the crystals is P21/b; the unit-cell dimensions are a = 11.169, b = 21.535, c = 7.863 A, alpha = beta = 90 degrees, gamma = 122.45 degrees, Z = 4. The structure is solved by direct methods, and refined with the full-matrix least-squares procedure. The data on the structure of the pyrrol carboxamide backbone allow the following conclusions to be made about the molecular structure of the distamycin type antibiotics: (i) the amide groups have normal trans-configuration with slightly shortened C-C and C-N bonds adjacent to the pyrrol rings, (ii) the N-methyl groups of the pyrrol rings and the oxygen atoms of the amide groups have the same orientation with respect to the backbone. In the distamycin A analog molecule the pyrrol rings and amide group between them are approximately coplanar.

Chemical Phenomena↗
PubMed1979

Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA AT pairs.

Interaction of DNA with the analogs of the antibiotic distamycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The binding isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to poly(dA).poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GC pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA.

Adenine↗
PubMed1977

Distamycin A and its analogs as agents for blocking of endo R. EcoRI activity.

Distamycin A (Dst) and its analogs protect the lambda phage DNA from cleavage with endoR. EcoRI and show selective affinity for different recognition sites of endoR. EcoRI on this DNA producing enlarged DNA fragments of various composition and length. The affinity of the antibiotic for DNA is influenced by the number of pyrrol carboxamide units in Dst molecule and does not strongly depend on the substitution of the N-methyl group by the N-propyl one. Since in the complex with DNA the antibiotics of the Dst type are localized in its minor groove a conclusion can be made that the minor groove of DNA is needed for the interaction of the restriction endonuclease with DNA.

Coliphages↗
PubMed1976

A code controlling specific binding of regulatory proteins to DNA.

A possible code is suggested that describes a correspondence between amino acid sequences in stereospecific sites of regulatory proteins and nucleotide sequences at the control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet with single-stranded regions at the ends of the beta-structure. The binding reaction between regulatory protein and double-helical DNA is accompanied by significant structural alterations at stereospecific sites of the protein and DNA. Half of the hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. The code states a correspondence between four amino acid residues at a stereospecific site of the regulatory protein and an AT (GC) base pair at the control site. It predicts that there are six fundamental amino acid residues (serine, threonine, histidine, asparagine, glutamine and cysteine) whose arrangement in the stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially.

Amino Acid Sequence↗
PubMed1976

A model for the binding of lac repressor to the lac operator.

A model is suggested for the lac repressor binding to the lac operator in which the repressor polypeptide chain sequences from Gly 14 to Ala 32 and from Ala 53 to Leu 71 are involved in specific interaction with operator DNA. A correspondence between the protein and DNA sequences is found which explains specificity of the repressor binding to the lac operator. The model can be extended to describe specific binding of other regulatory proteins to DNA.

Amino Acid Sequence↗