共查询到20条相似文献,搜索用时 31 毫秒
1.
Zheleznaya LA Perevyazova TA Zheleznyakova EN Matvienko NI 《Biochemistry. Biokhimii?a》2002,67(4):498-502
A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55°C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA–DNA duplexes and therefore cannot be used for detection of RNA targets. 相似文献
2.
Interaction of clinically important human DNA topoisomerase I poison, topotecan, with double-stranded DNA 总被引:2,自引:0,他引:2
Streltsov S Oleinikov V Ermishov M Mochalov K Sukhanova A Nechipurenko Y Grokhovsky S Zhuze A Pluot M Nabiev I 《Biopolymers》2003,72(6):442-454
Topotecan (TPT), a water-soluble derivative of camptothecin, is a potent antitumor poison of human DNA topoisomerase I (top1) that stabilizes the cleavage complex between the enzyme and DNA. The role of the recently discovered TPT affinity to DNA remains to be defined. The aim of this work is to clarify the molecular mechanisms of the TPT-DNA interaction and to propose the models of TPT-DNA complexes in solution in the absence of top1. It is shown that TPT molecules form dimers with a dimerization constant of (4.0 +/- 0.7) x 10(3) M(-1) and the presence of DNA provokes more than a 400-fold increase of the effective dimerization constant. Flow linear dichroism spectroscopy accompanied by circular dichroism, fluorescence, and surface-enhanced Raman scattering experiments provide evidence that TPT dimers are able to bind DNA by bridging different DNA molecules or distant DNA structural domains. This effect may provoke modification of the intrinsic geometry of the cruciform DNA structures, leading to the appearance of new crossover points that serve as the sites of the top1 loading position. The data presume the hypothesis of TPT-mediated modulation of top1-DNA recognition before ternary complex formation. 相似文献
3.
Kerrigan JE Pilch DS Ruchelman AL Zhou N Liu A Liu L LaVoie EJ 《Bioorganic & medicinal chemistry letters》2003,13(20):3395-3399
In this paper, we present our results from a docking study of the title compounds with the DNA/topoisomerase I complex based on the recently published X-ray crystal structure of the topotecan/DNA/topoisomerase I ternary cleavable complex (Staker, B.L., et al. PNAS 2002, 99, 15387) using the Autodock program. Simple intermolecular docking energies (E(dock)) correlate well with in vitro DNA cleavage data suggesting that the binding mode from the crystal structure is a reasonable binding mode for these compounds. 相似文献
4.
Chad S. Braun Gouri S. Jas Sirirat Choosakoonkriang Gary S. Koe Janet G. Smith C. Russell Middaugh 《Biophysical journal》2003,84(2):1114-1123
The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed. 相似文献
5.
6.
Sangita Kachhap Pragya Priyadarshini 《Journal of biomolecular structure & dynamics》2017,35(6):1153-1167
Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5′-(T/C)TAATTAA(T/A)(T/A)G-3′ DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein–DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll–3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll–3A01–Al. In addition, this change in secondary structure of Cll does not allow it to form protein–protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al–3A01–Cll where Cll binds to Al–3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein–protein interactions while proposing likely mechanism of recognition in ternary complex, Al–Cll–DNA. 相似文献
7.
Paola B Arimondo Alexandre Boutorine Brigitte Baldeyrou Christian Bailly Masayasu Kuwahara Sidney M Hecht Jian-Sheng Sun Thérèse Garestier Claude Hélène 《The Journal of biological chemistry》2002,277(5):3132-3140
To achieve a sequence-specific DNA cleavage by topoisomerase I, derivatives of the antitumor drug camptothecin have been covalently linked to triple helix-forming oligonucleotides that bind in a sequence-specific manner to the major groove of double-helical DNA. Triplex formation at the target sequence positions the drug selectively at the triplex site, thereby stimulating topoisomerase I-mediated DNA cleavage at this site. In a continuous effort to optimize this strategy, a broad set of conjugates consisting of (i) 16-20-base-long oligonucleotides, (ii) alkyl linkers of variable length, and (iii) camptothecin derivatives substituted on the A or B quinoline ring were designed and synthesized. Analysis of the cleavage sites at nucleotide resolution reveals that the specificity and efficacy of cleavage depends markedly on the length of both the triple-helical structure and the linker between the oligonucleotide and the poison. The optimized hybrid molecules induced strong and highly specific cleavage at a site adjacent to the triplex. Furthermore, the drug-stabilized DNA-topoisomerase I cleavage complexes were shown to be more resistant to salt-induced reversal than the complexes induced by camptothecin alone. Such rationally designed camptothecin conjugates could provide useful antitumor drugs directed selectively against genes bearing the targeted triplex binding site. In addition, they represent a powerful tool to probe the molecular interactions in the DNA-topoisomerase I complex. 相似文献
8.
Human polynucleotide kinase (hPNK), which possesses both 5'-DNA kinase and 3'-DNA phosphatase activities, is a DNA repair enzyme required for processing and rejoining of single- and double-strand-break termini. Full-length hPNK was subjected to sedimentation and spectroscopic analyses in association with its ligands, a 20-mer oligonucleotide, ATP, and AMP-PNP (a nonhydrolyzable analogue of ATP). Sedimentation equilibrium measurements indicated that hPNK was a monomer in the presence and absence of the ligands. Circular dichroism measurements revealed that the ligands induced different conformational changes in hPNK, although AMP-PNP induced the same conformational changes as ATP. CD also indicated that the oligonucleotide could bind to the protein-AMP-PNP complex. Protein-ligand binding affinities and stoichiometries were determined by measuring changes in protein intrinsic fluorescence. Titrating hPNK with the oligonucleotide indicated tight binding with a K(d) value of 1.3 microM and with 1:1 stoichiometry. A 5'-phosphorylated oligonucleotide with the same sequence exhibited an almost 6-fold lower affinity (K(d) value, 7.2 microM). ATP and AMP-PNP bound with high affinity (K(d) values, respectively, of 1.4 and 1.6 microM), and the observed binding stoichiometries were 1:1. Furthermore, the nonphosphorylated oligonucleotide was able to bind to hPNK in the presence of AMP-PNP with a K(d) value of 2.5 microM, confirming the formation of a ternary complex. This study provides the first direct physical evidence for such a ternary complex involving a polynucleotide kinase, AMP-PNP, and an oligonucleotide, and supports a reaction mechanism in which ATP and DNA bind simultaneously to the enzyme. 相似文献
9.
KorB is a member of the ParB family of bacterial partitioning proteins. The protein encoded by the conjugative plasmid RP4 is part of the global control circuit and regulates the expression of plasmid genes, the products of which are involved in replication, transfer, and stable inheritance. KorB is a homodimeric protein which binds to palindromic 13 bp DNA sequences [5'-TTTAGC((G)/(C))GCTAAA-3'] present 12 times in the 60 kb plasmid. Each KorB subunit is composed of two domains; the C-domain is responsible for the dimerization of the protein, whereas the N-terminal domain recognizes and binds to the operator sequence (O(B)). Here we describe results of a Raman spectroscopic study of the interaction of the N-domain with a double-stranded model oligonucleotide composed of the palindromic binding sequence and terminal 5'-A(Br)U and AG-3' bases. Comparison of the Raman spectra of the free KorB N-domain and O(B) DNA with the spectrum of the complex reveals large differences. KorB-N binds in the major groove of the O(B) DNA, and the interactions induce changes in the DNA backbone and in the secondary structure of the protein. 相似文献
10.
P B Arimondo C Bailly A S Boutorine P Moreau M Prudhomme J S Sun T Garestier C Hélène 《Bioconjugate chemistry》2001,12(4):501-509
Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity. 相似文献
11.
Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance in archeons. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermophilic archeon Sulfolobus. Structures of several complexes of Sso7d/Sac7d with DNA octamers are known. These structures are characterized by sequence unspecific minor groove binding of the proteins and sharp kinking of the double helix. Corresponding Raman vibrational signatures have been identified in this study. A Raman spectroscopic analysis of Sac7d binding to the oligonucleotide decamer d(GAGGCGCCTC)(2) reveals large conformational perturbations in the DNA structure upon complex formation. Perturbed Raman bands are associated with the vibrational modes of the sugar phosphate backbone and frequency shifts of bands assigned to nucleoside vibrations. Large changes in the DNA backbone and partial B- to A-form DNA transitions are indicated that are closely associated with C2'-endo/anti to C3'-endo/anti conversion of the deoxyadenosyl moiety upon Sac7d binding. The major spectral feature of Sac7d binding is kinking of the DNA. Raman markers of minor groove binding do not largely contribute to spectral differences; however, clear indications for minor groove binding come from G-N2 and G-N3 signals that are supported by Trp24 features. Trp24 is the only tryptophan present in Sac7d and binds to guanine N3, as has been demonstrated clearly in X-ray structures of Sac7d-DNA complexes. No changes of the Sac7d secondary structure have been detected upon DNA binding. 相似文献
12.
Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)-brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)-brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)-brazilin complex and DNA bases. 相似文献
13.
Boukhalfa-Heniche FZ Hernández B Gaillard S Coïc YM Huynh-Dinh T Lecouvey M Seksek O Ghomi M 《Biopolymers》2004,73(6):727-734
Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C. 相似文献
14.
Collisions between replication forks and topoisomerase-drug-DNA ternary complexes result in the inhibition of DNA replication and the conversion of the normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes inhibited the strand displacement activities of these DNA helicases. Unlike replication fork arrest, however, this general inhibition of DNA helicases by Topo IV-norfloxacin-DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases. UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not sufficient to generate cytotoxic DNA lesion. 相似文献
15.
The solution structure of the complex formed between an oligonucleotide containing a two-base bulge (5'-CACGCAGTTCGGAC.5'-GTCCGATGCGTG) and DDI, a designed synthetic agent, has been elucidated using high-resolution NMR spectroscopy and restrained molecular dynamic simulation. DDI, which has been found to modulate DNA strand slippage synthesis by DNA polymerase I [Kappen, L. S., Xi, Z., Jones, G. B., and Goldberg, I. H. (2003) Biochemistry 42, 2166-2173], is a wedge-shaped spirocyclic molecule whose aglycone structure closely resembles that of the natural product, NCSi-gb, which strongly binds to an oligonucleotide containing a two-base bulge. Changes in chemical shifts of the DNA upon complex formation and intermolecular NOEs between DDI and the bulged DNA duplex indicate that agent specifically binds to the bulge site of DNA. The benzindanone moiety of DDI intercalates via the minor groove into the G7-T8-T9.A20 pocket, which consists of a helical base pair and two unpaired bulge bases, stacking with the G7 and A20 bases. On the other hand, the dihydronaphthalenone and aminoglycoside moieties are positioned in the minor groove. The aminoglycoside, which is attached to spirocyclic ring, aligns along the A20T21G22 sequence of the nonbulged strand, while the dihydronaphthalenone, which is restrained by the spirocyclic structure, is positioned near the G7-T8-T9 bulge site. The aminoglycoside is closely aligned with the dihydronaphthalenone, preventing its intercalation into the bulge site. In the complex, the unpaired purine (G7) is intrahelical and stacks with the intercalating moiety of DDI, whereas the unpaired pyrimidine (T8) is extrahelical. The structure of the complex formed by binding of the synthetic agent to the two-base bulged DNA reveals a binding mode that differs in important details from that of the natural product, explaining the different binding specificity for the bulge sites of DNA. The structure of the DDI-bulged DNA complex provides insight into the structure-binding affinity relationship, providing a rational basis for the design of specific, high-affinity probes of the role of bulged nucleic acid structures in various biological processes. 相似文献
16.
Oxidative destruction of DNA by the adriamycin-iron complex 总被引:7,自引:0,他引:7
The 2:1 adriamycin-Fe(III) complex is able to bind to DNA and to catalyze its oxidative destruction. The binding of the drug-metal complex to DNA is indicated by characteristic spectral changes which are different from those seen with adriamycin intercalation and by the propensity of the drug-metal complex to precipitate DNA. Furthermore, intercalated adriamycin appears not to be available for iron binding. The resulting ternary complex is quite stable: it is not disrupted by incubation in the presence of EDTA and can be isolated by using Sephadex G-50 column chromatography. Disruption of the ternary complex requires vigorous conditions (extraction with phenol at 60 degrees C). The adriamycin-iron complex in free solution has the capacity to catalyze the reduction of oxygen by thiols. The DNA-bound drug-metal complex preserves this capacity over a wide range of complex/DNA ratios. As a consequence of this thiol-dependent oxygen reduction, DNA is cleaved. This thiol-dependent DNA cleavage has been shown to require hydrogen peroxide as an intermediate product. These results have led us to propose that the thiol-dependent DNA cleavage reaction has two stages involving (1) reduction of oxygen leading to hydrogen peroxide and then (2) peroxide-dependent DNA cleavage. An unusual property of this reaction is that the cleavage is not random but gives rise to a defined 2300 base pair fragment. 相似文献
17.
Bleomycin (Blm) is an antitumor agent which binds to specific sequences of DNA and as HO(2)-Fe(III)Blm causes single and double strand cleavage. In the present investigation, binding of O(2)-Co(II)Blm to a native DNA polymer, calf thymus DNA, was examined using conventional Raman spectroscopy. O(2)-Co(II)Blm is a model for O(2)-Fe(II)Blm, the direct precursor of HO(2)-Fe(III)Blm. Although the DNA polymer retained a predominant B-form structure, Raman spectral evidence was obtained for localized structural changes to A, C and Z-DNA forms. The presence of these alternate DNA forms within B-DNA implied the presence of B/A, B/C and B/Z junctions. The observed changes in DNA secondary structure were attributed to perturbation of structural water resulting from binding of O(2)-Co(II)Blm within the minor groove. 相似文献
18.
19.
Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC)and two 7-mer oligonucleotide conjugates,one of which was modified on its 3'-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group,and the other one was linked through its 5'-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD)spectra were detected for all possible oligonucleotide and/or conjugate combinations.In addition,molecular modeling simulation of the complex structure was carried out.It was found that CD spectra did not show serious changes in the B-helix structure of the duplex.The interaction between perylene-and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner,but azido-group located on the side of DNA chain minor groove. 相似文献