Subramaniam G, Paz M M, Suresh Kumar G, Das A, Palom Y, Clement C C, Patel D J, Tomasz M
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, City University of New York, New York, New York 10021, USA.
Biochemistry. 2001 Sep 4;40(35):10473-84. doi: 10.1021/bi010965a.
2,7-Diaminomitosene (2,7-DAM), the major metabolite of the antitumor antibiotic mitomycin C, forms DNA adducts in tumor cells. 2,7-DAM was reacted with the deoxyoligonucleotide d(GTGGTATACCAC) under reductive alkylation conditions. The resulting DNA adduct was characterized as d(G-T-G-[M]G-T-A-T-A-C-C-A-C) (5), where [M]G stands for a covalently modified guanine, linked at its N7-position to C10 of the mitosene. The adducted oligonucleotide complements with itself, retaining 2-fold symmetry in the 2:1 drug-duplex complex, and provides well-resolved NMR spectra, amenable for structure determination. Adduction at the N7-position of G4 ([M]G, 4) is characterized by a downfield shift of the G4(H8) proton and separate resonances for G4(NH(2)) protons. We assigned the exchangeable and nonexchangeable proton resonances of the mitosene and the deoxyoligonucleotide in adduct duplex 5 and identified intermolecular proton-proton NOEs necessary for structural characterization. Molecular dynamics computations guided by 126 intramolecular and 48 intermolecular distance restraints were performed to define the solution structure of the 2,7-DAM-DNA complex 5. A total of 12 structures were computed which exhibited pairwise rmsd values in the 0.54-1.42 A range. The 2,7-DAM molecule is anchored in the major groove of DNA by its C10 covalently linked to G4(N7) and is oriented 3' to the adducted guanine. The presence of 2,7-DAM in the major groove does not alter the overall B-DNA helical structure. Alignment in the major groove is a novel feature of the complexation of 2,7-DAM with DNA; other known major groove alkylators such as aflatoxin, possessing aromatic structural elements, form intercalated complexes. Thermal stability properties of the 2,7-DAM-DNA complex 5 were characteristic of nonintercalating guanine-N7 alkylating agents. Marked sequence selectivity of the alkylation by 2,7-DAM was observed, using a series of oligonucleotides incorporating variations of the 5'-TGGN sequence as substrates. The selectivity correlated with the sequence specificity of the negative molecular electrostatic potential of the major groove, suggesting that the alkylation selectivity of 2,7-DAM is determined by sequence-specific variation of the reactivity of the DNA. The unusual, major groove-aligned structure of the adduct 5 may account for the low cytotoxicity of 2,7-DAM.
2,7-二氨基丝裂霉素（2,7-DAM）是抗肿瘤抗生素丝裂霉素C的主要代谢产物，可在肿瘤细胞中形成DNA加合物。在还原烷基化条件下，使2,7-DAM与脱氧寡核苷酸d(GTGGTATACCAC)发生反应。所得的DNA加合物被表征为d(G-T-G-[M]G-T-A-T-A-C-C-A-C)（5），其中[M]G代表共价修饰的鸟嘌呤，其N7位与丝裂霉素的C10相连。加合的寡核苷酸可自身互补，在2:1的药物-双链体复合物中保持2倍对称性，并提供分辨率良好的核磁共振谱，适用于结构测定。G4（[M]G，4）的N7位加合的特征是G4(H8)质子的化学位移向低场移动以及G4(NH(2))质子的单独共振。我们对加合物双链体5中丝裂霉素和脱氧寡核苷酸的可交换和不可交换质子共振进行了归属，并确定了结构表征所需的分子间质子-质子核Overhauser效应（NOE）。在126个分子内和48个分子间距离限制的指导下进行了分子动力学计算，以确定2,7-DAM-DNA复合物5的溶液结构。总共计算了12个结构，其两两之间的均方根偏差（rmsd）值在0.54 - 1.42 Å范围内。2,7-DAM分子通过其与G4(N7)共价连接的C10锚定在DNA的大沟中，并在加合的鸟嘌呤的3'方向上定向。大沟中2,7-DAM的存在不会改变整体的B-DNA螺旋结构。在大沟中的排列是2,7-DAM与DNA络合的一个新特征；其他已知的大沟烷基化剂，如具有芳香结构元件的黄曲霉毒素，形成插入复合物。2,7-DAM-DNA复合物5的热稳定性特性是非插入性鸟嘌呤-N7烷基化剂的特征。使用一系列含有5'-TGGN序列变体的寡核苷酸作为底物，观察到2,7-DAM烷基化具有明显的序列选择性。这种选择性与大沟负分子静电势的序列特异性相关，表明2,7-DAM的烷基化选择性由DNA反应性的序列特异性变化决定。加合物5不寻常的大沟排列结构可能解释了2,7-DAM的低细胞毒性。
