Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.3 (topoisomerase)
 9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1H- and 31P-NMR and UV-absorption studies were carried out with the oligonucleotide strands d(AGCT-TATC-ATC-GATAAGCT) (-ATC-) and d(AGCTTATC-GAT-GATAAGCT) (-GAT-) contained in the strongest and salt resistant cleavage site for topoisomerase II in pBR322 DNA. We found that the two oligonucleotides were stabilized under a hairpin structure characterized by a eight base pair stem and a three base loop at low DNA and salt concentrations. In such experimental conditions, only the -GAT- oligonucleotide displayed a partial homoduplex structure in slow equilibrium with its folded structure. Temperature dependencies of imino protons showed that the partial homoduplex of -GAT- melted at a lower temperature than the hairpin structure. It was suggested that the appearance of the partial homoduplex in -GAT- is related to the formation of two stabilizing (G.T) mismatched base pairs in the central loop of this structure. Finally, it was inferred from the dispersion of chemical shifts in the 31P-NMR spectra that the distortions affecting the backbone of the hairpin loop are larger in the case of -ATC- compared with -GAT-. At the same time NOEs proved that the base stacking was stronger within the loop of the -ATC- hairpin.
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PMID:Hairpins in a DNA site for topoisomerase II studied by 1H- and 31P-NMR. 747 27

We report on the structural study of the single-stranded 19mer oligonucleotide d(AGCTTATC-ATC-GATAAGCT) 22(+). This corresponds to the 15-to-33(+) strand of pBR322 DNA belonging to a strong cleavage site (site 22) for topoisomerase II coupled to antitumor drugs VP-16 or ellipticine. The partially self-complementary nature of this oligonucleotide makes likely its folding into a hairpin structure. To assess this property we carried out a quantitative analysis based on joint calculations and NMR experiments. The latter required two-dimensional (NOESY, P-COSY, TOCSY and proton-detected 1H-31P), and three-dimensional (NOESY-TOCSY) spectra to achieve the assignment of the overcrowded sugar H4' ad H5'/H5" proton region. For molecular modeling, the JUMNA program was used together with NMR constraints; namely, the distances and the backbone torsion angles provided by NOEs and homo- and heteronuclear coupling constants. Experimental results proved that the 19mer oligonucleotide adopted a stable hairpin structure characterized by an eight base-pair stem and a three-membered loop (central-ATC-segment). Homonuclear 1H-1H and heteronuclear 1H-31P coupling constant measurements provided information on the conformational heterogeneity of the sugar and phosphate groups within both the stem and the loop. Restrained energy minimizations starting with different structures resulted in a family of closely related structures. All low-energy molecules presented the same, rather compact, folded structure with the base-stacking continuing into the loop, a sharp turn occurring between residues T10 and C11, and strong backbone distortions at the loop-stem junction.
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PMID:The hairpin structure of a topoisomerase II site DNA strand analyzed by combined NMR and energy minimization methods. 894 75

The structural analysis of two single-stranded DNAs d(AGCTTATCATCGATAAGCT) (ATC-19) and d(AGCTTATCGATGATAAGCT) (GAT-19) was performed by NMR and restrained molecular dynamics. These oligonucleotides reproduce the 15-33 segment of phage pBR322 DNA, which contains a strong cleavage site for topoisomerase II coupled to the antitumor drugs VP-16 and ellipticine. Because of their partial palindromic nature, the two oligonucleotides ATC-19 and GAT-19 may fold back into stable hairpin structures, consisting of a stem of eight base-pairs and a loop of three residues. NMR assignments and conformational parameters were determined from combined 2D NOESY, COSY and 1H-31P spectra. Conformations of ATC-19 and GAT-19 hairpins were calculated using the X-PLOR 3.1 program. Structures were generated through simulated annealing procedures starting from 50 structures with randomized torsion angles. A good convergence was observed for ATC-19 molecules, while no consensus was found for GAT-19. Within the GAT-19 loop, the base stacking was poor and no hydrogen bond could be detected. In contrast, ATC-19 displayed a well-defined three residue loop stabilized by both extensive base stackings and hydrogen bonding between the N3 atom of the adenine ring and the amino group of the cytosine ring. The results confirm our earlier ATC-19 structure obtained by a completely different calculation procedure (JUMNA) and the higher thermal stability of ATC-19 compared to GAT-19. Moreover, due to its mismatched base-pair, the ATC-19 loop may be better described as a single residue loop rather than a three residue loop. Comparison of this loop to those containing sheared purine.purine base-pairs revealed striking resemblances, particularly on the backbone angle combination. Finally, the differences observed between the ATC-19 and GAT-19 structures could help toward understanding the sequential cleavage of DNA strands by topoisomerase II.
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PMID:Comparative structural analysis by [1H,31P]-NMR and restrained molecular dynamics of two DNA hairpins from a strong DNA topoisomerase II cleavage site. 978 73

Our previous NMR and modeling studies have shown that the single-stranded 19mer oligonucleotides d(AGCTTATC-ATC-GATAA GCT) -ATC- and d(AGCTTATC-GAT-GATAAGCT) -GAT- encompassing the strongest topoisomerase II cleavage site in pBR322 DNA could form stable hairpin structures. A new sheared base-pair, the pyrimidine-purine C x A, was found to close the single base -ATC- loop, while -GAT- displayed a flexible loop of three/five residues with no stabilizing interactions. Now we report a structural study on -GAC-, an analog of -GAT-, derived through the substitution of the loop residue T by C. The results obtained from NMR, non-denaturing PAGE, UV-melting, circular dichroism experiments and restrained molecular dynamics indicate that -GAC- adopts a hairpin structure folded through a single residue loop. In the -GAC- hairpin the direction of the G9 sugar is reversed relative to the C8 sugar, thus pushing the backbone of the loop into the major groove. The G9 x C11 base-pair closing the loop is thus neither a sheared base-pair nor a regular Watson-Crick one. Although G9 and C11 are paired through hydrogen bonds of Watson-Crick type, the base-pair is not planar but rather adopts a wedge-shaped geometry with the two bases stacked on top of each other in the minor groove. The distortion decreases the sugar C1'-C1' distance between the paired G9 and C11, to 8 A versus 11 A in the standard B-DNA. The A10 residue at the center of the loop interacts with the G9 x C11 base-pair, and seems to contribute to the extra thermal stability displayed by -GAC- compared to -GAT-. Test calculations allowed us to identify the experimental NOEs critical for inducing the distorted G.C Watson-Crick base-pair. The preference of -GAC- for a hairpin structure rather than a duplex is confirmed by the diffusion constant values obtained from pulse-field gradient NMR experiments. All together, the results illustrate the high degree of plasticity of single-stranded DNAs which can accommodate a variety of turn-loops to fold up on themselves.
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PMID:A DNA hairpin with a single residue loop closed by a strongly distorted Watson-Crick G x C base-pair. 1061 Jul 69

We carried out a structural study of the DNA heterochiral strand d (AGCTTATCAT(L)CGATAAGCT), -AT(L)C-, where T(L) (L thymine ) replaces T (natural D-thymine). -AT(L)C- is a structural analog of -ATC- that belongs to a strong topoisomerase II DNA cleavage site and which has been shown to resolve into a hairpin structure with a stem formed by eight Waston-Crick base-pairs and a single residue loop closed by an A.C sheared base-pair. Although - AT(L)C-, like its parent -ATC-, folds into a hairpin structure at low and high DNA concentrations it displays a lower stability (Tm of 56 degrees C versus 58.5 degrees C). Several NMR features in -AT(L)C- account for the disruption of the A.C pairing in the loop and a weakening of the C.G base-pair stability at the stem-loop junction. For instance, the exchange of the loop imino protons with solvent is accelerated compared with the natural oligonucleotide -ATC-. The higher flexibility of the heterochiral loop is confirmed by the results of NMR restrained molecular dynamics. In the calculated final structures of -AT(L)C-, the T10(L) residue moves the A9 and C11 residues away, thus preventing the loop closure through a C.A sheared base-pair and the achievement of a good base-base or sugar-base stacking. Actually, most of the stabilizing interactions present in -ATC- are lost in the heterochiral - AT(L)C- explaining its weaker stability.
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PMID:NMR study of a heterochiral DNA hairpin:impact of L-enantiomery in the loop. 1179 Jan 44

DNA topoisomerase II is an enzyme that specializes in DNA disentanglement. It catalyzes the interconversion of DNA between different topological states. This event requires the passage of one duplex through another one via a transient double-strand break. Topoisomerase II is able to process any type of DNA, including structures such as DNA juxtapositions (crossovers), DNA hairpins or cruciforms, which are recognized with high specificity. In this review, we focused our attention on topoisomerase II recognizing DNA substrates that possess particular geometries. A strong cleavage site, as we identified in pBR322 DNA in the presence of ellipticine (site 22), appears to be characterized by a cruciform structure formed from two stable hairpins. The same sequence could also constitute a four-way junction structure stabilized by interactions involving ATC sequences. The latter have been shown to be able to promote Holliday junctions. We reviewed the recent literature that deals with the preferential recognition of crossovers by various topoisomerases. The single molecule relaxation experiments have demonstrated the differential abilities of the topoisomerases to recognize crossovers. It appears that enzymes, which distinguish the chirality of the crossovers, possess specialized domains dedicated to this function. We also stress that the formation of crossovers is dependent on the presence of adequate stabilizing sequences. Investigation of the impact of such structures on enzyme activity is important in order to both improve our knowledge of the mechanism of action of the topoisomerase II and to develop new inhibitors of this enzyme.
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PMID:Does topoisomerase II specifically recognize and cleave hairpins, cruciforms and crossovers of DNA? 1739 86