Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III -: Direct identification of Lys-212 as the active nucleophilic residue
Purification and characterization of human NTH1, a homolog of Escherichia coli endonuclease III -: Direct identification of Lys-212 as the active nucleophilic residue
复制标题
DOI：
10.1074/jbc.273.34.21585
复制
发表时间：
1998-08-21
影响因子：
4.8
通讯作者：
Mitra, S
中科院分区：
文献类型：
--
作者：
Ikeda, S;Biswas, T;Mitra, S
人核酸内切酶III（hNTH 1）是大肠杆菌酶（Nth）的同系物，是一种具有脱碱基（脱嘌呤/脱嘧啶（AP））裂解酶活性的DNA糖基化酶，特异性切割DNA中氧化损伤的嘧啶。克隆了该酶的cDNA，并在大肠杆菌中以谷胱甘肽S-转移酶融合多肽的形式表达了全长酶（304个氨基酸残基）。杆菌具有两个额外氨基酸残基的纯化野生型蛋白质和在NH 1末端缺失22个残基的截短蛋白质具有相同的活性，并且在280和410 nm处具有最大吸收，后者是由于[4Fe-4S]簇的存在，如在E. coli Nth.该酶裂解含胸腺嘧啶二醇的I型质粒DNA和含二氢尿嘧啶（DHU）的寡核苷酸双链体。该蛋白质的摩尔消光系数为5.0 × 10（4），pi为10。以含DHU的寡核苷酸双链体作为底物，K-m为47 nM，k（cat）与0.6/min相似，与DHU是否与G或A配对无关。该酶进行p-消除，并在活性位点残基和碱去除后产生的脱氧核糖之间形成席夫碱。通过肽-寡核苷酸加合物的序列分析证实了Lys-212是酶的活性位点。然而，用Arg-212替换产生了比野生型蛋白低约85倍的催化特异性的活性酶。用hNTH 1进行的DNA酶I足迹分析显示，在受损链中以碱基损伤为中心的10个核苷酸和互补链中的15个核苷酸的延伸（G与5 '边界处的损伤相对）受到保护。免疫学研究表明，HeLa细胞包含一个单一的hNTH物种的预测大小，定位在细胞核和细胞质。
The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH, terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 x 10(4) and a pi of 10. With the DHU-containing oligonucleotide duplex as substrate, the K-m was 47 nM, and k(cat) was similar to 0.6/min, independent of whether DHU paired with G or A. The enzyme carries out p-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct, Furthermore, replacing Lys-212 with Gin inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5'-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.