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Journal of the American Chemical Society Jul 2006The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im),...
The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.
Topics: Base Pairing; DNA; Nucleic Acid Conformation; Nylons; Oligonucleotides; Protein Binding; Pyrroles
PubMed: 16834381
DOI: 10.1021/ja0621795 -
The Journal of Biological Chemistry May 1990We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of... (Comparative Study)
Comparative Study
We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A.T-rich DNA. Three highly conserved regions within each of the known HMG-I proteins is closely related to the consensus sequence T-P-K-R-P-R-G-R-P-K-K. A synthetic oligopeptide corresponding to this consensus "binding domain" (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin and to the dye Hoechst 33258. In vitro these ligands, which also preferentially bind to A.T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide also contains novel structural features such as a predicted Asx bend or "hook" at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or "bristles" which may contribute to the binding properties of the HMG-I proteins. The predicted BD peptide structure, which we refer to as the "A.T-hook," represents a previously undescribed DNA-binding motif capable of binding to the minor groove of stretches of A.T base pairs.
Topics: Adenine; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Bisbenzimidazole; Cattle; Computer Simulation; DNA; High Mobility Group Proteins; Humans; Interleukin-2; Mice; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Protein Binding; Sequence Homology, Nucleic Acid; Thymine
PubMed: 1692833
DOI: No ID Found -
Biophysical Journal Aug 2006We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios....
We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.
Topics: Animals; Biophysics; Cattle; Circular Dichroism; DNA; Indicators and Reagents; Ligands; Models, Chemical; Nylons; Protein Binding; Spectrophotometry; Thymus Gland
PubMed: 16679369
DOI: 10.1529/biophysj.105.080564 -
Annals of the New York Academy of... Mar 1977A cell culture system has been utilized to measure the effects of drugs on DNA synthesis in uninfected and HSV-(herpes simplex virus)-infected KB cells. DNA from...
A cell culture system has been utilized to measure the effects of drugs on DNA synthesis in uninfected and HSV-(herpes simplex virus)-infected KB cells. DNA from HSV-infected cells was separated into viral and cellular components by isopycnic centrifugation in CsCl gradients. The amount of [3H]thymidine incorporated into acid-insoluble material was measured in the absence and presence of drugs. Dose-response relationships were established by linearly regressing the probit value of the percent inhibition DNA synthesis against the logarithm of drug concentration. Fifty percent inhibitory (I50) concentrations were interpolated from the corresponding regression lines for inhibition of the following: (i) DNA synthesis is uninfected KB cells, (ii) total DNA synthesis in HSV-infected KB cells (iii) cellular DNA synthesis in HSV-infected cells, and (iv) viral DNA synthesis in HSV-infected cells. We have derived an index (SI, selective index) that quantifies the preferential inhibition of viral or uninfected cellular DNA synthesis. This index can be expressed as SI = log10 I50 concentration for DNA synthesis in uninfected cells divided by I50 concentration for viral DNA synthesis in HSV-infected cells. The SI is positive if viral DNA synthesis is inhibited preferentially and negative if uninfected cellular DNA synthesis is more strongly inhibited. A positive SI value of 0.5 was obtained for the clinically useful antiviral drug arabinosyladenine (ara-A) and a value of 0.4 for its metabolite, arabinosylhypoxanthine (ara-H). Although the adenosine deaminase inhibitor coformycin greatly increased the potency of ara-A, the inhibitor did not increase the selectivity of the drug (SI = 0.3). Stallimycin (distimycin A) (SI = 0.3) and phosphonoacetic acid (SI = 0.3) were similarly effective in preferentially inhibiting the synthesis of HSV DNA. In contrast, arabinosylcytosine (ara-C) and ribavirin inhibited DNA synthesis in uninfected cells to a greater degree than viral DNA synthesis (SI = -0.5 and -1.9, respectively). An analysis of the advantages and limitations of this experimental procedure is made and the suggestion is offered that the in vitro determination of a drug's selective index may be a valid predictor of clinical usefulness.
Topics: Antiviral Agents; Cells, Cultured; DNA; DNA, Viral; Simplexvirus; Virus Cultivation; Virus Replication
PubMed: 212984
DOI: 10.1111/j.1749-6632.1977.tb21976.x -
Nucleic Acids Research Nov 1996By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This...
By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin. This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species. This protein was found to be expressed both in cancer and normal tissues. By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa. We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4.
Topics: Animals; Antineoplastic Agents; Binding Sites; Bisbenzimidazole; Cell Line; Cricetinae; DNA; DNA Damage; DNA-Binding Proteins; Distamycins; Electrophoresis, Polyacrylamide Gel; Humans; Intercalating Agents; Jurkat Cells; Melphalan; Mice; Mice, Inbred C57BL; Nitrogen Mustard Compounds; Tumor Cells, Cultured
PubMed: 8932377
DOI: 10.1093/nar/24.21.4227 -
European Journal of Biochemistry Mar 1972
Binding of analogues of the antibiotics distamycin A and netropsin to native DNA. Effect of chomophore systems and basic residues of the oligopeptides on thermal stability, conformation and template activity of the DNA complexes.
Topics: Amidines; Anti-Bacterial Agents; Cell-Free System; Chemical Phenomena; Chemistry; Circular Dichroism; DNA; Drug Stability; Guanidines; Hot Temperature; Nucleic Acid Conformation; Nucleic Acid Denaturation; Peptides; Phosphorus Isotopes; Protein Binding; Pyrroles; RNA; Spectrum Analysis; Templates, Genetic; Ultraviolet Rays
PubMed: 5043330
DOI: 10.1111/j.1432-1033.1972.tb01742.x -
American Journal of Human Genetics Mar 1984Both aneuploid cells in mitosis and nondisjunction in meiosis increase with advancing age. The cause(s) of these phenomena remains unknown. Here, it was confirmed that a... (Comparative Study)
Comparative Study
Both aneuploid cells in mitosis and nondisjunction in meiosis increase with advancing age. The cause(s) of these phenomena remains unknown. Here, it was confirmed that a positive Cd-band reflects the presence of a functioning centromere while a negative reaction is indicative of its inactivation or loss. We applied the Cd-banding technique to mitotic spreads obtained from 14 aged and 13 control females (peripheral blood culture). Of 6,474 scored chromosomes from the aged women, 62(0.96%) were Cd-negative; this was the case in 12 of 3,861 (0.31%) chromosomes from the younger controls. The difference was highly significant (P less than .001). About 60% of the 62 Cd-negative chromosomes from aged women belong to the C group. Chromosomes from six of the 14 aged females were additionally examined by either the C-band or the distamycin-DAPI technique; of 2,080 chromosomes, 13 (0.63%) showed premature separation of their centromeres (control: 7/5, 080, 0.14%). Our findings suggest that in aged women, the chromosomes tend to lose their Cd-positive material and the function of the centromere.
Topics: Aged; Aging; Aneuploidy; Centromere; Chromosome Banding; Chromosome Deletion; Chromosomes; Chromosomes, Human, 6-12 and X; Female; Humans; Karyotyping; Male; Sex Factors
PubMed: 6711561
DOI: No ID Found -
PloS One 2011Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts...
BACKGROUND
Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts as a transcriptional modulator and exerts a key role in viral DNA replication. Thus, E2 constitutes an attractive target for developing antiviral agents. E2 is a homodimeric protein that interacts with the DNA target through an α-helix of each monomer. However, a peptide corresponding to the DNA recognition helix of HPV-16 E2 binds DNA with lower affinity than its full-length DNA binding domain. Therefore, in an attempt to promote the DNA binding of the isolated peptide, we have designed a conjugate compound of the E2 α-helix peptide and a derivative of the antibiotic distamycin, which involves simultaneous minor- and major-groove interactions.
METHODOLOGY/PRINCIPAL FINDINGS
An E2 α-helix peptide-distamycin conjugate was designed and synthesized. It was characterized by NMR and CD spectroscopy, and its DNA binding properties were investigated by CD, DNA melting and gel shift experiments. The coupling of E2 peptide with distamycin does not affect its structural properties. The conjugate improves significantly the affinity of the peptide for specific DNA. In addition, stoichiometric amounts of specific DNA increase meaningfully the helical population of the peptide. The conjugate enhances the DNA binding constant 50-fold, maintaining its specificity.
CONCLUSIONS/SIGNIFICANCE
These results demonstrate that peptide-distamycin conjugates are a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and suggest that a bipartite major/minor groove binding scaffold can be a useful approach for therapeutic treatment of HPV infection.
Topics: Amino Acid Sequence; Antiviral Agents; Base Sequence; Biomimetic Materials; DNA, Viral; DNA-Binding Proteins; Distamycins; Human papillomavirus 16; Models, Molecular; Molecular Sequence Data; Oncogene Proteins, Viral; Papillomavirus Infections; Peptide Fragments; Protein Structure, Secondary; Pyrroles; Substrate Specificity
PubMed: 21799846
DOI: 10.1371/journal.pone.0022409 -
The Journal of Biological Chemistry Sep 1990The therapeutic and cytotoxic effects exerted by DNA-binding drugs used for chemotherapy originate from a rather large variety of modifications sustained by the nucleic...
The therapeutic and cytotoxic effects exerted by DNA-binding drugs used for chemotherapy originate from a rather large variety of modifications sustained by the nucleic acids upon interaction with the chemical agents. Notably, these modifications are generally considered as involving the following localized chemical or structural processes: base alkylations, frameshift mutations or strand breakages at specific sites, interstrand cross-links, and local structural transitions within the secondary configurations. We find that antitumor agents hinder or prevent altogether the long range packaging of DNA molecules into compact, ordered states. This effect, observed even at low drug to base pair ratios, is general: it is induced by DNA groove binders as well as by intercalators. Nucleoprotein complexes are found to be efficiently protected against the decondensing effect of the drugs. These observations point toward a generic mechanism for the effectiveness of DNA-binding drugs against tumor cells and viruses as well as for the severe effects of chemotherapy on male fertility: actively dividing systems, such as tumor cells, are characterized by regions of chromatin which are decondensed for the purpose of replication and transcription, and therefore accessible to the drugs. Similarly, both viral infection and spermatogenesis, where histones are replaced by protamines, involve transient formation of relatively uncondensed DNA species and subsequent packaging into extremely tight structures.
Topics: Chromomycins; Circular Dichroism; DNA; Dactinomycin; Daunorubicin; Distamycins; Magnesium; Microscopy, Electron; Nucleic Acid Conformation; Pyrroles; Spectrometry, Fluorescence
PubMed: 2394757
DOI: No ID Found -
Proceedings of the National Academy of... Nov 1996DNA molecules containing inosine in place of guanosine and/or 2,6-diaminopurine in place of adenine have been synthesized and tested as substrates for binding of... (Comparative Study)
Comparative Study
PCR-based development of DNA substrates containing modified bases: an efficient system for investigating the role of the exocyclic groups in chemical and structural recognition by minor groove binding drugs and proteins.
DNA molecules containing inosine in place of guanosine and/or 2,6-diaminopurine in place of adenine have been synthesized and tested as substrates for binding of sequence-selective ligands, both small and large. Footprinting patterns reveal that the binding sites for AT- or GC-specific antibiotics (distamycin or mithramycin, respectively) are completely changed in the modified DNAs, as expected for direct sequence readout involving contact with the purine 2-amino group. However, we also find large changes in the binding of HMG-D, a member of the HMG-1 family of chromosomal proteins, pointing to an indirect influence of the exocyclic amino group on ligand binding via an effect on the deformability of the double helix. This interpretation is confirmed by the finding that deoxyuridine-containing poly- and oligonucleotides, which lack the exocyclic methyl group of thymidine in the major groove, interact 5-10 times more strongly with HMG-D than do their counterparts containing natural nucleotides.
Topics: 2-Aminopurine; Base Composition; Base Sequence; Binding Sites; DNA; DNA Footprinting; DNA Primers; Deoxyribonuclease I; Distamycins; Guanosine; High Mobility Group Proteins; Hydrogen Bonding; Inosine; Molecular Sequence Data; Nucleic Acid Conformation; Plasmids; Plicamycin; Polymerase Chain Reaction
PubMed: 8942984
DOI: 10.1073/pnas.93.24.13623