Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 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

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