Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.
Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.
复制标题
DOI：
10.1073/pnas.93.11.5197
复制
发表时间：
1996-05
影响因子：
11.1
通讯作者：
Patricia Auffret;van der;KEMPt;Dominique THOMASt;Reigine Barbeyi;Regina DE OLIVEIRAt;Serge BOITEUXt
中科院分区：
文献类型：
--
作者：
Patricia Auffret;van der;KEMPt;Dominique THOMASt;Reigine Barbeyi;Regina DE OLIVEIRAt;Serge BOITEUXt
利用大肠杆菌自发突变株(FPG Muy)克隆了酿酒酵母OGG1基因，该基因编码7，8-二氢-8-氧鸟嘌呤(8-OxoG)的DNA糖基酶活性。用酵母DNA文库转化大肠杆菌(FPG Muy)，筛选出自发诱变能力较弱的克隆。抗突变活性与pYSB10-11-KBP重组载体有关。含有pYSB10的大肠杆菌无细胞提取物(Fpg Muy)具有切割一个34聚体寡核苷酸的酶活性，该寡核苷酸含有一个8-oxoG相对胞嘧啶(8-oxoG/C)。对抑制自发突变和过量产生8-OxoG/C裂解活性的1.7kbp的酵母DNA片段进行了测序，并将其定位在第13号染色体上。DNA测序确定了一个开放阅读框，命名为OGG1，编码一个376个氨基酸的蛋白质，相对分子质量为43 kDa。将OGG1基因插入到pUC19中，得到pYSB110。用含有pYSB110的大肠杆菌(FPG)对酿酒酵母Ogg1蛋白进行纯化，得到明显均一的蛋白。Ogg1蛋白具有释放8-oxoG和2，6-diamino-4-hydroxy-5-N-methylformamidopyrimidine.的脱氧核糖基酶活性Ogg1蛋白优先切割含有8-OxoG相对胞嘧啶(8-OxoG/C)或胸腺嘧啶(8-OxoG/T)的DNA。相反，Ogg1蛋白不会切割腺嘌呤与8-OxoG(8-OxoG/A)相对的双链。Ogg1蛋白切割单链的机制可能是由于8-oxoG被切除，随后在脱嘌呤/脱嘧啶酶切位点发生了β-消除。
A spontaneous mutator strain of Escherichia coli (fpg mutY) was used to clone the OGG1 gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7,8-dihydro-8-oxoguanine (8-OxoG). E. coli (fpg mutY) was transformed by a yeast DNA library, and clones that showed a reduced spontaneous mutagenesis were selected. The antimutator activity was associated with pYSB10, an 11-kbp recombinant plasmid. Cell-free extracts of E. coli (fpg mutY) harboring pYSB10 possess an enzymatic activity that cleaves a 34-mer oligonucleotide containing a single 8-oxoG opposite a cytosine (8-OxoG/C). The yeast DNA fragment of 1.7 kbp that suppresses spontaneous mutagenesis and overproduces the 8-OxoG/C cleavage activity was sequenced and mapped to chromosome XIII. DNA sequencing identified an open reading frame, designated OGG1, which encodes a protein of 376 amino acids with a molecular mass of 43 kDa. The OGG1 gene was inserted in plasmid pUC19, yielding pYSB110. E. coli (fpg) harboring pYSB110 was used to purify the Ogg1 protein of S. cerevisiae to apparent homogeneity. The Ogg1 protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. The Ogg1 protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Ogg1 protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Ogg1 protein is probably due to the excision of 8-OxoG followed by a beta-elimination at the resulting apurinic/apyrimidinic site.