Chan C L, Wu Z, Ciardelli T, Eastman A, Bresnick E
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
Arch Biochem Biophys. 1993 Jan;300(1):193-200. doi: 10.1006/abbi.1993.1027.
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis, and carcinogenesis. Recombinant human MGMT was isolated from an Escherichia coli high performance expression system and purified to homogeneity. The kinetic and DNA-binding properties of the recombinant human MGMT were studied. The purified human MGMT reacted stoichiometrically with methylated DNA under second-order rate kinetics. The rate constant with normal methylated DNA was 1 x 10(9) M-1 min-1 at 37 degrees C. The binding to DNA was the rate determining step in the repair process. Approximately eight base pairs of the DNA substrate were covered by the human MGMT protein. The affinity constant for interaction of DNA to MGMT was approximately 4.7 x 10(5) M-1. The binding to methylated DNA was also examined; the binding affinity to methylated DNA was two times higher than that to unmodified DNA. The interaction with DNA induced a conformational change in the human MGMT protein as monitored by circular dichroism and fluorescence analysis. A similar conformational change was induced by both methylated and unmodified DNA.
O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）是一种DNA修复蛋白，在化疗、诱变和致癌过程中发挥重要作用。重组人MGMT从大肠杆菌高效表达系统中分离出来并纯化至同质。对重组人MGMT的动力学和DNA结合特性进行了研究。纯化的人MGMT在二级速率动力学下与甲基化DNA发生化学计量反应。在37℃时，与正常甲基化DNA的速率常数为1×10⁹ M⁻¹ min⁻¹。与DNA的结合是修复过程中的速率决定步骤。人MGMT蛋白覆盖了大约八个碱基对的DNA底物。DNA与MGMT相互作用的亲和常数约为4.7×10⁵ M⁻¹。还检测了与甲基化DNA的结合；对甲基化DNA的结合亲和力比对未修饰DNA的高两倍。通过圆二色性和荧光分析监测，与DNA的相互作用诱导了人MGMT蛋白的构象变化。甲基化和未修饰的DNA均诱导了类似的构象变化。
