Mauffret O, Amir-Aslani A, Maroun R G, Monnot M, Lescot E, Fermandjian S
Départment de Biologie Structurale et de Pharmacologie Moléculaire (CNRS UMR 1772), PR2, Institut Gustave Roussy, Villejuif Cedex, 94805, France.
J Mol Biol. 1998 Oct 30;283(3):643-55. doi: 10.1006/jmbi.1998.2095.
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.
通过核磁共振（NMR）和受限分子动力学对两条单链DNA d(AGCTTATCATCGATAAGCT)（ATC - 19）和d(AGCTTATCGATGATAAGCT)（GAT - 19）进行了结构分析。这些寡核苷酸复制了噬菌体pBR322 DNA的15 - 33片段，该片段含有与抗肿瘤药物VP - 16和玫瑰树碱偶联的拓扑异构酶II的强切割位点。由于它们部分的回文性质，两条寡核苷酸ATC - 19和GAT - 19可能会折叠成稳定的发夹结构，由一个八个碱基对的茎和一个三个残基的环组成。通过二维NOESY、COSY和1H - 31P谱的组合确定了NMR归属和构象参数。使用X - PLOR 3.1程序计算了ATC - 19和GAT - 19发夹的构象。从50个具有随机扭转角的结构开始，通过模拟退火程序生成结构。观察到ATC - 19分子有良好的收敛性，而GAT - 19未达成共识。在GAT - 19环内，碱基堆积较差，未检测到氢键。相比之下，ATC - 19展示了一个明确的三个残基环，该环通过广泛的碱基堆积以及腺嘌呤环的N3原子与胞嘧啶环的氨基之间的氢键而稳定。结果证实了我们早期通过完全不同的计算程序（JUMNA）获得的ATC - 19结构，以及ATC - 19相对于GAT - 19具有更高的热稳定性。此外，由于其错配碱基对，ATC - 19环可能更好地被描述为一个单残基环而非三个残基环。将该环与含有剪切嘌呤 - 嘌呤碱基对的环进行比较，发现了显著的相似性，特别是在主链角度组合方面。最后，在ATC - 19和GAT - 19结构之间观察到的差异可能有助于理解拓扑异构酶II对DNA链的顺序切割。
