The hairpin structure of a topoisomerase II site DNA strand analyzed by combined NMR and energy minimization methods

被引:16
作者
AmirAslani, A [1 ]
Mauffret, O [1 ]
Sourgen, F [1 ]
Neplaz, S [1 ]
Maroun, RG [1 ]
Lescot, E [1 ]
Tevanian, G [1 ]
Fermandjian, S [1 ]
机构
[1] INST GUSTAVE ROUSSY,DEPT BIOL & PHARMACOL STRUCT PHARMACOL MOL,CNRS URA 147,F-94805 VILLEJUIF,FRANCE
关键词
DNA hairpin; loop; topoisomerase II; NMR; energy minimizations;
D O I
10.1006/jmbi.1996.0615
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 H-1-P-31), 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 H-1-H-1 and heteronuclear H-1-P-31 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. (C) 1996 Academic Press Limited
引用
收藏
页码:776 / 788
页数:13
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