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Molecular structure of the netropsin-d(CGCGATATCGCG) complex: DNA conformation in an alternating AT segment 总被引:17,自引:0,他引:17
The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step. 相似文献
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Specific interaction of netropsin, distamycin-3 and analogs with LC duplexes: reversion towards the B form of the 2''-deoxy-.2''-deoxy-2''-fluoro-hybrid duplexes upon specific interaction with netropsin, distamycin-3 and analogs. 总被引:2,自引:2,他引:0
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Binding of the B-form specific ligands netropsin and distamycin-3, -4 and -5 has been used to monitor the presence and/or the inducibility of a B-type structure in various poly-inosinic.poly-cytidilic double stranded polymers with deoxyribose, ribose or 2'-deoxy-2'-fluororibose as sugar on either strand. The efficiency of binding was followed by circular dichroism and further evaluated by the increase in melting temperature of the complexes. The efficient binding of netropsin and distamycins to the hybrid polymer (dIfl)n. (dC)n demonstrated that the fluorine carrying strand may undergo a A to B-type transition reflecting a change of the 2'-deoxy-2'-fluororibose from the 3'-endo to the 1'-exo or 2'-endo pucker. The less efficient binding of the same ligands to the reverse hybrid (dI)n.(dCfl)n showed that the geometry of the pyrimidine strand is the most critical for the specific interaction. Taking into account the recent findings about the regular hydration in the minor groove of the B-type dodecamer dCGCGAATTCGCG in solid-state, the different binding modes observed between the different polymers and antibiotics are explained by differences in their possibilities of hydration. Binding of netropsin to a double stranded deoxypolymer is interpreted as a local replacement of water molecules by netropsin in the minor groove hydration network which is typical of the B-form. 相似文献
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R B Macgregor 《Analytical biochemistry》1992,204(2):324-327
Hydroxyl radicals yield footprints of DNA-ligand interactions that are uniform in intensity and display single base pair resolution. It is shown here that brief illumination of dilute aqueous solutions of hydrogen peroxide with a standard uv transilluminator can be used to generate hydroxyl radicals for footprinting studies. Photogenerated hydroxyl radicals are used to footprint netropsin, a drug that interacts with the minor groove of DNA. The method presented eliminates two of the reagents used in conventional Fenton-reaction-based hydroxyl radical footprinting. It has the further advantage that the extent of cleavage of the DNA can be precisely regulated by controlling the illumination time. Because light is used to drive the reaction, photogenerated hydroxyl radicals can be used to footprint DNA-ligand interactions under experimental conditions of temperature and pressure inaccessible to Fenton-reaction chemistry. 相似文献
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D J Patel 《European journal of biochemistry》1979,99(2):369-378
The structure of the netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex (one antibiotic molecule/self-complementary octanucleodide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson-Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA . dT-containing tetranucleotide segment dA-dA-dT-dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen-bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C-6 and adenine C-8 protons. The close proximity of the pyrrole rings of the antibiotic and the base-pair edges in the minor groove is manifested in the downfield shifts (0.3--0.5 ppm) of the pyrrole C-3 protons of netropsin and one adenine C-2 proton and one thymine N-3 base-pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid. 相似文献
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DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini.
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In recent years two mechanisms have been proposed for the production of DNA strand breaks in cells undergoing oxidative stress: (i) DNA attack by OH radical, produced by Fenton reaction catalyzed by DNA-bound iron; and (ii) DNA attack by calcium-activated nucleases, due to the increase of cytosolic and nuclear calcium induced by oxidative stress. We set out to investigate the participation of the former mechanism by detecting and quantifying 3'-phosphoglycolate, a 3' DNA terminus known to be formed by OH radical attack to the deoxyribose moiety, followed by sugar ring rupture and DNA strand rupture. These structures were found in DNA of monkey kidney cells exposed to hydrogen peroxide, iron nitrilotriacetate or ascorbate, all species known to favor a cellular pro-oxidant status. The method employed to measure 3' phosphoglycolate was the 32P-postlabeling assay. Repair time course experiments showed that it takes 10 h for 3'-phosphoglycolate to be removed from DNA. It was found that the DNA of both control cells and cells exposed to hydrogen peroxide had a very poor capacity of supporting in vitro DNA synthesis, catalyzed by DNA polymerase I. If the DNA was previously incubated with exonuclease III, an enzyme able to expose 3'-OH primers by removal of 3'-phosphoglycolate and 3'-phosphate termini the in vitro synthesis was substantially increased. This result shows that either of these termini are present at the break and that 3'-hydroxyl termini are virtually absent. At least 25% of the strand breaks exhibited 3'-phosphoglycolate termini as determined by the 32P-postlabeling assay, but due to the characteristic of the method this percentage is likely to be higher. These results favor the hypothesis that an oxidative agent generated by Fenton reaction is responsible for DNA strand breakage in cells undergoing oxidative stress. 相似文献
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T Shiraki M Uesugi Y Sugiura 《Biochemical and biophysical research communications》1992,188(2):584-589
Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin. 相似文献
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Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA. 相似文献
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L S Kappen I H Goldberg B L Frank L Worth D F Christner J W Kozarich J Stubbe 《Biochemistry》1991,30(8):2034-2042
The thiol-activated neocarzinostatin chromophore cleaves duplex oligonucleotides containing the sequence-TGTTTGA-, producing 3'-phosphoglycolate and 3'-phosphate fragments at T, indicating the involvement of 4'- as well as 5'-chemistry at this residue. Substitution of deuterium for hydrogen at the C-4' position of the affected T leads to a kinetic isotope effect (kH/kD) of 4.0 on the formation of the glycolate-ended product, whereas deuterium at C-5' of the same T reveals kH/kD of 1.6 in the formation of the phosphate-ended product. The proportion of the products representing 4'- and 5'-chemistry can be shifted on the basis of isotope selection effects. A second product resulting from 4'-chemistry, the abasic site associated with 4'-hydroxylation, has been identified as an alkali-labile site, and as a pyridazine derivative formed after cleavage by hydrazine. A comparable isotope effect on its production (kH/kD = 3.7) relative to that of 3'-phosphoglycolate production is consistent with a common intermediate, a putative 4'-peroxy radical, in their formation. The formation of both products of 4'-chemistry is oxygen-dependent, and the internal partitioning between them (3'-phosphate or 3'-phosphoglycolate) is influenced by thiols. Moreover, the nitroaromatic radiation sensitizer misonidazole can substitute for dioxygen, yielding 3'-phosphoglycolate and alkali-labile 3'-phosphate ends, indicative of 4'-chemistry. In addition to the internal partitioning of 4'-chemistry, thiols also affect the overall extent of cleavage (4' plus 5') and the relative partitioning between both sites of attack (4' or 5'). 相似文献
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A molecular mechanics and molecular dynamics approach was used to examine the structure of complexes formed between the d(CGCGAATTCGCG)2 duplex and netropsin, distamycin, and four carbocyclic analogues of netropsin and distamycin (1-4). The resulting structures of the ligand-DNA model complexes and their energetics were examined. It is predicted that the compounds 1-4 should have a decreased affinity for the minor groove of AT-rich regions in comparison to netropsin and distamycin. From the energetic analysis it appears that van der Waals and electrostatic interactions are more important than specific hydrogen bonds in stabilizing the ligand-duplex complexes. We predict that compounds 1 and 2 are effectively isohelical with the DNA minor groove. The superior DNA-binding afforded by 1 and 2 in comparison to 3 and 4 results from their more effective penetration into the minor groove and smaller perturbation of molecular structure upon complex formation. 相似文献
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N Patel H Berglund L Nilsson R Rigler L W McLaughlin A Gr?slund 《European journal of biochemistry》1992,203(3):361-366
Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10(5) M-1 at 25 degrees C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as delta G = -29 kJ/mol, delta H = -12 kJ/mol and delta S = +55 J.mol-1.K-1 at 25 degrees C. These parameters resemble those of the interaction of poly[(dG-dC).(dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence. 相似文献
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S L Grokhovski? V A Nikolaev V E Zubarev A N Surovaia A L Zhuze B K Chernov N Iu Sidorova A S Zasedatelev G V Gurski? 《Molekuliarnaia biologiia》1992,26(6):1274-1297
Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove. 相似文献
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Binding of a Hoechst dye to d(CGCGATATCGCG) and its influence on the conformation of the DNA fragment 总被引:15,自引:0,他引:15
M A Carrondo M Coll J Aymami A H Wang G A van der Marel J H van Boom A Rich 《Biochemistry》1989,28(19):7849-7859
Hoechst dye 33258 is a planar drug molecule that binds to the minor groove of DNA, especially where there are a number of A.T base pairs. We have solved the structure of the Hoechst dye bound to the DNA dodecamer d(CGCGATATCGCG) at 2.3 A. This structure is compared to that of the same dodecamer with the minor-groove-binding drug netropsin bound to it, as well as to structures that have been solved for this Hoechst dye bound to a DNA dodecamer containing the central four base pairs with the sequence AATT. We find that the position of the Hoechst drug in this dodecamer is quite different from that found in the other dodecamer since it has an opposite orientation compared to the other two structures. The drug covers three of the four A.T base pairs and extends its piperazine ring to the first G.C base pair adjacent to the alternating AT segment. Furthermore, the drug binding has modified the structure of the DNA dodecamer. Other DNA dodecamers with alternating AT sequences show an alternation in the size of the helical twist between the ApT step (small twist) and the TpA step (large twist). In this structure the alternation is reversed with larger twists in the ApT steps than in the TpA step. In addition, there is a rotation of one of the thymine bases in the DNA dodecamer that is associated with hydrogen bonding to the Hoechst drug. This structure illustrates the considerable plasticity found in the DNA molecule when it binds to different planar molecules inserted into the minor groove. 相似文献
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Based on the finding that the wobble G.T mismatch 5' to the C of AGC.GCT results in switching of the attack chemistry by neocarzinostatin chromophore (NCS-Chrom) on the deoxyribose moiety of C from C-1' to C-4' [Kappen, L. S. & Goldberg, I. H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6706-6710], a series of mismatches has been explored for their effect on the chemistry of damage at the T of AGT.ACT in oligodeoxynucleotides, a site at which 4'-chemistry ordinarily occurs. Placement of a G.T mispair 5' to the T results in a marked increase in 4'-chemistry, as measured by the formation of breaks with 3'-phosphoglycolate ends and abasic sites due to 4'-hydroxylation. Strikingly, 4'-chemistry is induced at the T on the complementary strand, a site ordinarily restricted to 5'-chemistry. Substitution of dioxygen by the radiation sensitizer misonidazole exerts a pronounced effect on the partitioning of the 4'-chemistry in favor of the 3'-phosphoglycolate product. Both stable T.G and unstable T.C mismatches at the attack site itself are associated with marked inhibition of damage at this site. Whereas placement of the relatively stable G.A mismatch on the 5'-side of the T residue (AGT) results in substantial inhibition of damage at the T without shifting of chemistry, the same mismatch at the 3'-side of the attack site decreases damage only slightly but is associated with the appearance of significant 1'-chemistry. By contrast, no shift in chemistry is found with bleomycin, which attacks at C-4'.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution 总被引:3,自引:0,他引:3
We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. 相似文献
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Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas. 总被引:16,自引:5,他引:11
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A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini. 相似文献
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