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Bioscience, Biotechnology, and... May 2001The interaction of distamycin with ColE1 DNA was examined by using differential scanning calorimetry (DSC) taking the helix-coil transition theory of DNA into...
The interaction of distamycin with ColE1 DNA was examined by using differential scanning calorimetry (DSC) taking the helix-coil transition theory of DNA into consideration. Our results here strongly indicate that the affinity of distamycin to DNA, at a low distamycin concentration, depends highly on the DNA sequence, and preferential binding occurs to the sites of four to six successive A-T pairs having two or more successive G-C pairs on both their ends.
Topics: Bacteriocin Plasmids; Binding Sites; Calorimetry, Differential Scanning; DNA; Distamycins
PubMed: 11440155
DOI: 10.1271/bbb.65.1261 -
The Journal of Biological Chemistry Aug 2001Human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) terminates plus-strand DNA synthesis at the center of the HIV-1 genome, a process important for...
Human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) terminates plus-strand DNA synthesis at the center of the HIV-1 genome, a process important for HIV-1 infectivity. The central termination sequence contains two termination sites (Ter1 and Ter2) located at the 3'-end of A(n)T(m) motifs, and the narrowing of the DNA minor groove generated by these motifs is responsible for termination. Kinetic data associated with the binding of RT and its ability to elongate in vitro various DNA duplexes and triplexes surrounding the Ter2 terminator were analyzed using a simple kinetic scheme. At Ter2, RT still displays a reasonable affinity for the corresponding DNA, but the binding of the next nucleotide and above all its incorporation rate are markedly hampered. Features affecting the width of the minor groove act directly at this last step. The constraint exerted against elongation by the A(n)T(m) tract persists at two positions downstream of the terminator.
Topics: Base Sequence; DNA, Viral; Distamycins; HIV Reverse Transcriptase; Heparin; Kinetics; Molecular Sequence Data
PubMed: 11402036
DOI: 10.1074/jbc.M102974200 -
Genes & Development Mar 2001The alpha subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of...
The alpha subunit of E. coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers. We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin. Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding. On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs. Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding. These studies greatly improve the resolution of our picture of the promoter-RNAP interaction.
Topics: Amino Acid Motifs; Base Sequence; Binding Sites; DNA Footprinting; DNA, Bacterial; DNA-Directed RNA Polymerases; Distamycins; Escherichia coli; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Promoter Regions, Genetic; Protein Binding; Protein Subunits
PubMed: 11238372
DOI: 10.1101/gad.870001 -
Nucleic Acids Research Dec 2000Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase active on DNA substrates containing A/G, A/8-oxoG, A/C or G/8-oxoG mismatches. A truncated form of...
Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase active on DNA substrates containing A/G, A/8-oxoG, A/C or G/8-oxoG mismatches. A truncated form of MutY (M25, residues 1-226) retains catalytic activity; however, the C-terminal domain of MutY is required for specific binding to the 8-oxoG and is critical for mutation avoidance of oxidative damage. Using alkylation interference experiments, the determinants of the truncated and intact MutY were compared on A/8-oxoG-containing DNA. Several purines within the proximity of mismatched A/8-oxoG show differential contact by the truncated and intact MutY. Most importantly, methylation at the N7 position of the mismatched 8-oxoG and the N3 position of mismatched A interfere with intact MutY but not with M25 binding. The electrostatic contacts of MutY and M25 with the A/8-oxoG-containing DNA substrates are drastically different as shown by ethylation interference experiments. Five consecutive phosphate groups surrounding the 8-oxoG (one on the 3' side and four on the 5' side) interact with MutY but not with M25. The activities of the truncated and intact MutY are modulated differently by two minor groove-binding drugs, distamycin A and Hoechst 33258. Both distamycin A and Hoechst 33258 can inhibit, to a similar extent, the binding and glycosylase activities of MutY and M25 on A/G mismatch. However, binding and glycosylase activities on A/8-oxoG mismatch of intact MutY are inhibited to a lesser degree than those of M25. Overall, these results suggest that the C-terminal domain of MutY specifies additional contact sites on A/GO-containing DNA that are not found in MutY-A/G and M25-A/8-oxoG interactions.
Topics: Alkylation; Base Sequence; Bisbenzimidazole; Catalytic Domain; DNA; DNA Damage; DNA Glycosylases; DNA Methylation; DNA Repair; Distamycins; Dose-Response Relationship, Drug; Guanine; Mutation; N-Glycosyl Hydrolases; Nucleic Acid Conformation; Protein Binding
PubMed: 11095667
DOI: 10.1093/nar/28.23.4593 -
Molecular and Cellular Biology Oct 2000SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a...
SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.
Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Cell Line; Chromatin; Chromomycins; Cloning, Molecular; Conserved Sequence; DNA; DNA-Binding Proteins; Distamycins; Humans; Molecular Sequence Data; Mutation; Nuclear Proteins; Oligodeoxyribonucleotides; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Transfection
PubMed: 11003645
DOI: 10.1128/MCB.20.20.7480-7489.2000 -
Genes & Genetic Systems Jun 2000Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a...
Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.
Topics: Asian People; Base Sequence; Cell Line; Chromosome Fragile Sites; Chromosome Fragility; DNA, Complementary; Gene Expression; Heterozygote; Humans; Japan; Minisatellite Repeats; Molecular Sequence Data; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 10984839
DOI: 10.1266/ggs.75.149 -
The Journal of Biological Chemistry Nov 2000The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P...
The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5'-RCGG downward arrowCW-3' sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5'-CTATTGCGG downward arrowC-3' sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P(i). H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PDeltaN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Bacillus subtilis; Bacteriophages; Base Sequence; DNA; DNA, Superhelical; DNA-Binding Proteins; Distamycins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endodeoxyribonucleases; Endonucleases; Escherichia coli; Hydrolysis; Kinetics; Models, Biological; Models, Genetic; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Time Factors
PubMed: 10930407
DOI: 10.1074/jbc.M004309200 -
Proceedings of the National Academy of... Jul 2000Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side...
Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamycin binding reaction has not been studied in either of its binding modes, because of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant increase in fluorescence amplitude, accompanied by a small red shift, on binding distamycin to its specific target sites. This signal can be used to monitor drug binding in steady-state and time-resolved processes. Distamycin shows extremely fast association with the 1:1 binding site, with a bimolecular rate of 7 x 10(7) M(-1) small middle dots(-1) and also fairly rapid dissociation ( approximately 3 s(-1)). When DNA is in excess, there is a slow component in the association reaction whose rate decreases strongly with increasing DNA concentration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecular rate comparable to that at the 1:1 site, followed by a slow ( approximately 4 s(-1)) equilibration to the final population. Dissociation from the 2:1 site is approximately 40-fold slower than from the 1:1 site. This study provides the groundwork for analysis of the binding kinetics of longer polyamides and covalently linked polyamides that have recently been shown to inhibit transcription in vivo.
Topics: Anti-Bacterial Agents; Binding Sites; DNA; Diffusion; Distamycins; Flow Injection Analysis; Kinetics; Spectrometry, Fluorescence
PubMed: 10884413
DOI: 10.1073/pnas.97.14.7814 -
Nucleic Acids Research Jun 2000Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome,...
Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund-Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K(i) =1 microM). The distinct inhibition of WRN and BLM helicases by the minor groove binders suggest that these helicases unwind double-stranded DNA by a related mechanism.
Topics: Adenosine Triphosphatases; Bloom Syndrome; DNA Helicases; Distamycins; Enzyme Inhibitors; Exodeoxyribonucleases; Humans; Intercalating Agents; Kinetics; Ligands; Netropsin; RecQ Helicases; Recombinant Proteins; Topoisomerase I Inhibitors; Werner Syndrome; Werner Syndrome Helicase
PubMed: 10871376
DOI: 10.1093/nar/28.12.2420 -
Proceedings of the National Academy of... Jun 2000Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from the tunicate Ecteinascidia turbinata currently under phase II clinical trials for its...
Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from the tunicate Ecteinascidia turbinata currently under phase II clinical trials for its potent anticancer activity. ET-743 binds DNA in the minor groove and forms covalent adducts with some sequence specificity. It selectively inhibits in vitro binding of the CCAAT box factor NF-Y. In this study, we assayed ET-743 function in vivo on the HSP70 promoter. On heat induction, the drug blocks transcription rapidly at pharmacological concentrations and in a CCAAT-dependent manner, whereas the activity of the CCAAT-less simian virus 40 promoter is not affected. The effect is exerted at the mRNA level. The distamycin-like alkylating tallimustine is inactive in these assays. Binding of NF-Y and of the heat-shock factor is normal in ET-743-treated cells. Run-on analysis of several endogenous genes further proves that the drug has rapid, profound, and selective negative effects on transcription. Thus, this marine-derived compound is a promoter-specific, transcription-interfering agent.
Topics: 3T3 Cells; Animals; Antineoplastic Agents, Alkylating; CCAAT-Enhancer-Binding Proteins; DNA; DNA-Binding Proteins; Dioxoles; HSP70 Heat-Shock Proteins; Isoquinolines; Mice; Promoter Regions, Genetic; Tetrahydroisoquinolines; Trabectedin; Transcriptional Activation
PubMed: 10841573
DOI: 10.1073/pnas.97.12.6780