Escherichia coli endonuclease VIII: Cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants
Escherichia coli endonuclease VIII: Cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants
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DOI：
10.1128/jb.179.11.3773-3782.1997
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发表时间：
1997-06-01
影响因子：
3.2
通讯作者：
Wallace, SS
中科院分区：
文献类型：
--
作者：
Jiang, DY;Hatahet, Z;Wallace, SS
大肠杆菌拥有两种具有重叠底物特异性的 DNA 糖基化酶/脱嘌呤裂合酶活性，即核酸内切酶 III 和核酸内切酶 VIII，可识别并去除 DNA 中的氧化嘧啶。核酸内切酶 III 由第 n 个基因编码。核酸内切酶 VIII 现已纯化至明显的同质性，并且基因 nei 已通过反向遗传学进行克隆。基因 nei 位于大肠杆菌染色体上的 16 分钟处，编码 263 个氨基酸的蛋白质，该蛋白质在 N 端和 C 端区域与 5 种细菌 Fpg 蛋白具有显着同源性。构建了 nei 部分缺失替换突变体，并通过基因组 PCR、活性分析和蛋白质印迹分析证实了 nei 缺失。 nth nei 双突变体对电离辐射和过氧化氢过敏，但不如缺乏碱基切除修复的突变体 (xth nfo) 敏感。单个 nth 突变体表现出野生型对 X 射线的敏感性，而 nei 突变体始终比野生型稍微更敏感。通过利福平正向突变测定，缺乏核酸内切酶 III 和 VIII 的双突变体表现出强烈的自发突变表型（约 20 倍）。与表现出弱突变体表型的第n个突变体相反，nei单突变体表现为野生型。
Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant,vas constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.