Paquet F, Pérez C, Leng M, Lancelot G, Malinge J M
Centre de Biophysique Moléculaire, CNRS, Orleans France.
J Biomol Struct Dyn. 1996 Aug;14(1):67-77. doi: 10.1080/07391102.1996.10508930.
A 10 base pairs double-stranded oligonucleotide with the sequence d(CCTCG.CTCTC). d(GAGAG.CGAGG) containing a single interstrand cross-link resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(G.C/G.C) site by a cis-diammineplatinum(II) residue was analyzed by 1H NMR spectroscopy. All the exchangeable and nonexchangeable protons resonance lines (except some H5'-H5") were assigned. NOESY spectra and chemical shifts indicated that the cross-linkage of the guanines of G.5 and G.6 induced extrahelicity of C5 and C6. Moreover, several unusual proximities were observed such as: (i) NOE cross-peaks between the H2'-H2" of G.5 or G.6 and the aromatic proton of their 5' neighbor C4 or A7 (ii) the absence of cross-peak for the steps G.5-C6, C6-T7 and C5-G4 (iii) a strong NOESY connectivity between H8(G.5) and H2(A7). All these data allowed us to describe the head to tail arrangement of the two cross-linked guanines as well as their stacking with flanking neighbor nucleotides (G.5 with T7.A7 base pair and G.6 with C4.G4 base pair). Using all the NOESY and TOCSY data (208 constraints), we have obtained a solution structure of the cross-linked duplex by using the NMR-constrained molecular mechanics program JUMNA. The reversal position of the two cross-linked guanines placed the cis-diammineplatinum(II) residue in the minor groove. The stacking of the two cross-linked guanines with the surrounding bases induced a bend of 40 degrees toward the minor groove. The locally left-helix formation, the extrusion of the cytosines and the stacking of the platinated guanines led to an unwinding of 76 degrees. This value is in good agreement with the values deduced from gel electrophoresis experiments.
对一条10个碱基对的双链寡核苷酸d（CCTCG.CTCTC）.d（GAGAG.CGAGG）进行了¹H NMR光谱分析，该寡核苷酸在d（G.C/G.C）位点通过顺式二氨合铂（II）残基螯合DNA相反链上两个鸟嘌呤残基的N7位置形成了一个单链间交联。除了一些H5'-H5"外，所有可交换和不可交换质子的共振线都已归属。NOESY光谱和化学位移表明，G.5和G.6的鸟嘌呤交联诱导了C5和C6的螺旋外倾。此外，还观察到了几个不寻常的邻近关系，如：（i）G.5或G.6的H2'-H2"与其5'邻位C4或A7的芳香质子之间的NOE交叉峰；（ii）G.5-C6、C6-T7和C5-G4步的交叉峰缺失；（iii）H8（G.5）和H2（A7）之间有很强的NOESY连接性。所有这些数据使我们能够描述两个交联鸟嘌呤的头对尾排列方式以及它们与侧翼相邻核苷酸的堆积情况（G.5与T7.A7碱基对，G.6与C4.G4碱基对）。利用所有的NOESY和TOCSY数据（208个约束条件），我们通过使用NMR约束分子力学程序JUMNA获得了交联双链体的溶液结构。两个交联鸟嘌呤的反向位置使顺式二氨合铂（II）残基位于小沟中。两个交联鸟嘌呤与周围碱基的堆积导致向小沟方向弯曲40度。局部左螺旋的形成、胞嘧啶的挤出以及铂化鸟嘌呤的堆积导致解旋76度。这个值与凝胶电泳实验推断的值非常吻合。
