Asaeda A, Ide H, Asagoshi K, Matsuyama S, Tano K, Murakami A, Takamori Y, Kubo K
Department of Veterinary Radiology, School of Veterinary Medicine, College of Agriculture, Osaka Prefecture University, Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Biochemistry. 2000 Feb 29;39(8):1959-65. doi: 10.1021/bi9917075.
The activity of human methylpurine DNA N-glycosylase (hMPG) for major substrates was directly compared using two types of substrates, i.e., natural DNA and synthetic oligonucleotides. By the use of ARP assay detecting abasic sites in DNA, we first investigated the activity on the natural DNA substrates containing methylpurines, ethenopurines, or hypoxanthine (Hx) prepared by the conventional methods. After the treatment with hMPG, the amount of AP sites in methylated DNA was much higher than that in DNA containing ethenopurines or Hx. The oligodeoxynucleotide having a single 7-methylguanine (7-mG) was newly synthesized in addition to 1, N(6)-ethenoadenine (epsilonA)-, Hx-, and 8-oxoguanine-containing oligonucleotides. 7-mG was effectively excised by hMPG, though it might be less toxic than the other methylated bases with respect to mutagenesis and cell killing. The kinetic study demonstrated that k(cat)/K(m) ratios of the enzyme for epsilonA, Hx, and 7-mG were 2.5 x 10(-3), 1.4 x 10(-3), and 4 x 10(-4) min(-1) nM(-1), respectively. The oligonucleotides containing epsilonA effectively competed against 7-mG, while Hx substrates showed unexpectedly low competition. Concerning the effect of the base opposite damage, hMPG much preferred Hx.T to other Hx pairs, and epsilonA.C and epsilonA.A pairs were better substrates than epsilonA.T.
使用两种类型的底物，即天然DNA和合成寡核苷酸，直接比较了人甲基嘌呤DNA N-糖基化酶（hMPG）对主要底物的活性。通过使用ARP测定法检测DNA中的无碱基位点，我们首先研究了hMPG对通过常规方法制备的含有甲基嘌呤、乙烯嘌呤或次黄嘌呤（Hx）的天然DNA底物的活性。用hMPG处理后，甲基化DNA中的AP位点数量远高于含有乙烯嘌呤或Hx的DNA中的AP位点数量。除了含有1,N(6)-乙烯腺嘌呤（εA）、Hx和8-氧鸟嘌呤的寡核苷酸外，还新合成了含有单个7-甲基鸟嘌呤（7-mG）的寡脱氧核苷酸。hMPG能有效切除7-mG，尽管就诱变和细胞杀伤而言，它可能比其他甲基化碱基毒性更小。动力学研究表明，该酶对εA、Hx和7-mG的k(cat)/K(m)比值分别为2.5×10(-3)、1.4×10(-3)和4×10(-4) min(-1) nM(-1)。含有εA的寡核苷酸能有效与7-mG竞争，而Hx底物的竞争出乎意料地低。关于损伤碱基对面碱基的影响，hMPG对Hx.T的偏好远高于其他Hx对，并且εA.C和εA.A对比εA.T是更好的底物。
