Hashiguchi K, Stuart J A, de Souza-Pinto N C, Bohr V A
Laboratory of Molecular Gerontology, National Institute of Aging-IRP, National Institutes of Health, Box1, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.
Nucleic Acids Res. 2004 Oct 19;32(18):5596-608. doi: 10.1093/nar/gkh863. Print 2004.
The human Ogg1 glycosylase is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG) in both nuclear and mitochondrial DNA. Two distinct Ogg1 isoforms are present; alpha-Ogg1, which mainly localizes to the nucleus and beta-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from rho(0) cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that beta-Ogg1 protein levels are approximately 80% reduced in rho(0) cells, suggesting beta-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant beta-Ogg1. Surprisingly, recombinant beta-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since beta-Ogg1 lacks the C-terminal alphaO helix present in alpha-Ogg1, we generated mutant proteins with various amino acid substitutions in this domain. Of the seven amino acid positions substituted (317-323), we identified Val-317 as a novel critical residue for 8-oxoG binding and incision. Our results suggest that the alphaO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why beta-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of alpha-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that alpha-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. beta-Ogg1 may play a novel role in human mitochondria.
人类Ogg1糖基化酶负责修复核DNA和线粒体DNA中的8-氧代-7,8-二氢鸟嘌呤(8-氧代鸟嘌呤,8-oxoG)。存在两种不同的Ogg1亚型;α-Ogg1主要定位于细胞核,而β-Ogg1仅定位于线粒体。我们最近发现,缺乏线粒体DNA的ρ⁰细胞的线粒体具有与野生型细胞相似的8-氧代鸟嘌呤DNA糖基化酶活性。在此,我们表明ρ⁰细胞中β-Ogg1蛋白水平降低了约80%,这表明β-Ogg1不负责线粒体中8-氧代鸟嘌呤的切割。因此,我们对重组β-Ogg1的生化特性进行了表征。令人惊讶的是,重组β-Ogg1在体外未表现出任何显著的8-氧代鸟嘌呤DNA糖基化酶活性。由于β-Ogg1缺乏α-Ogg1中存在的C末端αO螺旋,我们在该结构域中生成了具有各种氨基酸取代的突变蛋白。在取代的七个氨基酸位置(317-323)中,我们确定缬氨酸-317是8-氧代鸟嘌呤结合和切割的新关键残基。我们的结果表明,αO螺旋对于8-氧代鸟嘌呤DNA糖基化酶活性绝对必要,因此其缺失可能解释了为什么β-Ogg1在体外不催化8-氧代鸟嘌呤的切割。蛋白质免疫印迹分析显示人类线粒体中存在大量α-Ogg1。结合先前的体内定位研究,这表明α-Ogg1蛋白可能为修复人类线粒体DNA中的这些损伤提供8-氧代鸟嘌呤DNA糖基化酶活性。β-Ogg1可能在人类线粒体中发挥新作用。