Comparison of substrate specificities of Escherichia coli endonuclease III and its mouse homologue (mNTH1) using defined oligonucleotide substrates
Comparison of substrate specificities of Escherichia coli endonuclease III and its mouse homologue (mNTH1) using defined oligonucleotide substrates
复制标题
DOI：
10.1021/bi000422l
复制
发表时间：
2000-09-19
期刊：
影响因子：
2.9
通讯作者：
Ide, H
中科院分区：
文献类型：
--
作者：
Asagoshi, K;Odawara, H;Ide, H
大肠杆菌核酸内切酶III（Endo III）及其同源物是活性氧和电离辐射引起的嘧啶损伤的主要修复酶。在本研究中，Endo III及其小鼠同源物（mNTH 1）的活动进行了比较，使用定义的寡核苷酸底物含有尿素残基（UR），两个顺式胸腺嘧啶乙二醇（TG）非对映异构体，5，6-二氢胸腺嘧啶（DHT），和5-羟基尿嘧啶（HOU）。将底物与Endo III和mNTH 1孵育，并基于通过凝胶电泳的产物分析比较它们的活性。Endo III识别所有测试的基底病变，但DHT的活性远低于其他底物。相比之下，尽管UR的一些偏好，mNTH 1表现出基本上相当的活动，包括DHT的所有基板。比较cis-TG和DHT的酶促参数显示，相对于cis-TG，亲和力（Km，27倍）和k（cat）（11倍）的大幅降低使得DHT成为Endo III的非常差的底物。mNTH 1对顺式-TG和DHT的亲和力和k（cat）相当，但mNTH 1的周转率（k（cat））明显低于Endo III。从反应机理的角度出发，考察了碱基配对效应对两种酶损伤识别的影响。Endo Ⅲ对UR和HOU的酶活性不依赖于碱基配对，而对顺式TG和DHT的酶活性在与G.对于mNTH 1，配对碱基效应仅对DHT明显。结合碱基切除修复酶提出的碱基翻转机制，讨论了配对碱基和酶的修复活性的变化。
Escherichia coli endonuclease III (Endo III) and its eukaryotic homologues are major repair enzymes for pyrimidine lesions formed by reactive oxygen species and ionizing radiation. In the present study, the activities of Endo III and its mouse homologue (mNTH1) have been compared using defined oligonucleotide substrates containing a urea residue (UR), two cis-thymine glycol (TG) diastereoisomers, 5,6-dihydrothymine (DHT), and 5-hydroxyuracil (HOU). The substrates were incubated with Endo III and mNTH1, and their activities were compared based on the product analysis by gel electrophoresis. Endo III recognized all base lesions tested, but the activity for DHT was extremely lower than other substrates. In contrast, albeit some preference of UR, mNTH1 showed essentially comparable activities for all substrates including DHT. Comparison of the enzymatic parameters for cis-TG and DHT revealed that large decreases in the affinity (K-m, 27-fold) and k(cat) (11-fold) relative to cis-TG made DHT an very poor substrate for Endo III. mNTH1 had comparable affinities and k(cat) for both cis-TG and DHT, though turnover (k(cat)) of mNTH1 was notably slower than Endo III. In view of the reaction mechanism, the paired base effect on the damage recognition by the two enzymes was also examined. The activities of Endo III for UR and HOU were paired base-independent, but those for cis TG and DHT were significantly enhanced when paired with G. With mNTH1, the paired base effect was evident only for DHT. The variations of the repair activity with paired bases and enzymes are discussed in relation to the base flipping mechanism suggested for base excision repair enzymes.