Somerville Lilla, Krynetski Eugene Y, Krynetskaia Natalia F, Beger Richard D, Zhang Weixing, Marhefka Craig A, Evans William E, Kriwacki Richard W
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
J Biol Chem. 2003 Jan 10;278(2):1005-11. doi: 10.1074/jbc.M204243200. Epub 2002 Oct 24.
Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest approximately 10 degrees opening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an approximately 80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the approximately 6 degrees C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.
巯嘌呤和硫鸟嘌呤是两种最广泛使用的抗白血病药物，它们通过作为脱氧-6-硫代鸟苷掺入DNA来发挥其细胞毒性和治疗作用。然而，这些硫嘌呤掺入DNA转化为细胞毒性的分子机制尚不清楚。本文展示的硫鸟嘌呤修饰的双链DNA的溶液结构表明，修饰对DNA结构的影响是微妙的，且局限于修饰的碱基对。具体而言，硫鸟嘌呤以酮式存在，与胞嘧啶形成减弱的沃森-克里克氢键，并导致修饰的碱基对向大沟方向适度张开约10度。相比之下，硫鸟嘌呤显著改变了碱基对动力学，与正常鸟嘌呤相比，与胞嘧啶的碱基对寿命降低了约80倍。这种扰动与修饰的寡核苷酸的DNA解链温度降低约6℃、硫鸟嘌呤亚氨基质子共振的1.13 ppm向上场位移以及硫鸟嘌呤亚氨基质子与水的交换速率大幅增加一致。我们的研究在DNA结构和动力学水平上为硫鸟嘌呤掺入DNA的影响提供了新的机制见解，为硫鸟嘌呤掺入对DNA加工酶活性的影响提供了解释，并为蛋白质对硫鸟嘌呤取代位点的特异性识别提供了分子基础。这些综合作用可能共同产生细胞反应，这些反应是硫嘌呤治疗作用的基础。
