Saccharomyces cerevisiae possesses two functional homologues of Escherichia coli endonuclease III.
Saccharomyces cerevisiae possesses two functional homologues of Escherichia coli endonuclease III.
复制标题
酿酒酵母拥有大肠杆菌核酸内切酶 III 的两种功能同源物。
DOI：
10.1021/bi973042h
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Doetsch,PW
中科院分区：
文献类型：
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作者：
You,HJ;Swanson,RL;Doetsch,PW
我们先前鉴定了两种不同的酵母氧自由基内切核酸酶基因（SCR 1和SCR 2），它们与大肠杆菌内切核酸酶III具有高度的序列相似性[Augeri，L.，李，Y。M.，巴顿，A. B.，和Doetsch，P. W.（1997）Biochemistry36，721 - 729]。SCR 1和SCR 2编码的蛋白在E.大肠杆菌，并纯化至表观均一。这两种蛋白质都能识别和切割含有二氢尿嘧啶、2，6-二氨基-4-羟基-5N-甲基甲酰胺基嘧啶（FaPy-7-MeGua）和脱碱基位点的DNA底物，但不能识别和切割含有尿嘧啶或8-氧代鸟嘌呤的DNA底物。纯化的Scr 2，而不是Scr 1，具有表明存在铁硫中心的光谱特性。Scr 1和Scr 2的动力学参数通过使用含有单个二氢尿嘧啶的寡核苷酸来确定。对Scr 1和Scr 2的推导氨基酸序列的分析表明，Scr 2具有铁硫基序，而Scr 1不具有该基序。然而，Scr 1具有长的带正电荷的N-末端，其可能是线粒体转运序列。SCR 1和SCR 2的靶向基因破坏产生了一个双突变体，该突变体对含二氢尿嘧啶的底物没有可检测的酶活性。北方印迹分析表明，甲萘醌诱导了SCR 1的表达，而对SCR 2无诱导作用。这些结果表明，虽然Scr 1和Scr 2都是E.大肠杆菌核酸内切酶III，它们彼此不同，就其氨基酸序列和诱导的DNA损伤剂，这表明它们的精确的生物学作用可能是不同的。
We previously identified two distinct genes ofSaccharomyces cerevisiaeredoxyendonuclease (SCR1andSCR2) which possess a high degree of sequence similarity toEscherichia coliendonuclease III [Augeri, L., Lee, Y. M., Barton, A. B., and Doetsch, P. W. (1997)Biochemistry36, 721−729]. The proteins encoded bySCR1andSCR2were overexpressed inE. coliand purified to apparent homogeneity. Both proteins recognized and cleaved DNA substrates containing dihydrouracil, 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine (FaPy-7-MeGua), and abasic sites but not DNA substrates containing uracil or 8-oxoguanine. Purified Scr2, but not Scr1, possesses spectral properties which indicate the presence of an iron−sulfur center. Kinetic parameters for Scr1 and Scr2 were determined by using an oligonucleotide containing a single dihydrouracil. Analysis of the deduced amino acid sequences of Scr1 and Scr2 suggests that Scr2 bears an iron−sulfur motif, while Scr1 does not have this motif. However, Scr1 has a long, positively charged N-terminus that could be a mitochondrial transit sequence. Targeted gene disruption ofSCR1andSCR2produced a double mutant that had no detectable enzymatic activity against the dihydrouracil-containing substrate. Northern blot analysis showed thatSCR1was induced by menadione, butSCR2was not. These results indicate that although Scr1 and Scr2 are both functional homologues ofE. coliendonuclease III, they differ from each other with respect to their amino acid sequences and inducibility by DNA damaging agents, suggesting that their precise biological roles may be different.