Lu R, Nash H M, Verdine G L
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Curr Biol. 1997 Jun 1;7(6):397-407. doi: 10.1016/s0960-9822(06)00187-4.
Guanine residues in the genome are vulnerable to attack by free radicals and reactive oxygen species. A major lesion thus produced, 8-oxoguanine (OG), causes mutations by mis-pairing with adenine during replication. In bacteria and budding yeast, OG is removed from the genome through the action of base-excision DNA repair (BER) enzymes, which catalyze expulsion of the aberrant base and excision of its sugar moiety from the DNA backbone. Although OG is known to be produced in and cleansed from mammalian genomes, the enzymes responsible for OG repair in these cells have remained elusive.
Here, we report the cloning and biochemical characterization of mammalian BER enzymes that specifically target OG residues in DNA. These 8-oxoguanine DNA glycosylases, hOgg1 (human) and mOgg1 (murine), are homologous to each other and to yeast Ogg1. They also contain an active site motif - the Helix-hairpin-Helix, Gly/Pro-rich-Asp motif - characteristic of a superfamily of BER proteins with a similar core fold and active site geometry. Both hOgg1 and mOgg1 exhibit exquisite selectivity for the base opposite OG in DNA, operating with high efficiency only on OG base-paired to cytosine. Furthermore, hOgg1 and mOgg1 are unable to process a panel of alternative lesions, including 8-oxoadenine, yet bind with high affinity to synthetic abasic site analogs. The proteins operate through a classical glycosylase/lyase catalytic mechanism; mutation of a catalytically essential lysine residue results in loss of catalytic potency but retention of binding to OG-containing oligonucleotides. The hOGG1 gene is localized on the short arm of chromosome 3 (3p25/26) in a region commonly deleted in cancers.
These results conclusively establish the existence and identity of an 8-oxoguanine DNA glycosylase/lyase in human and murine cells, completing the triad of proteins that together protect mammals from the genotoxic effects of guanine oxidation. The observation that at least one allele of hOGG1 is commonly deleted in cancer cells suggests that such cells may possess a reduced capacity to counter the mutagenic effects of reactive oxygen species, a deficiency that could increase their overall genomic instability. This speculation is fueled by recent observations that cells constitutively active for the Ras/Raf pathway constitutively produce high levels of superoxide, a known generator of OG.
基因组中的鸟嘌呤残基易受自由基和活性氧的攻击。由此产生的一种主要损伤产物8-氧代鸟嘌呤（OG），在复制过程中会与腺嘌呤错配从而导致突变。在细菌和芽殖酵母中，OG可通过碱基切除DNA修复（BER）酶的作用从基因组中去除，这些酶催化异常碱基的排出及其糖基部分从DNA主链上的切除。尽管已知OG在哺乳动物基因组中产生并被清除，但负责这些细胞中OG修复的酶一直难以确定。
在此，我们报告了特异性作用于DNA中OG残基的哺乳动物BER酶的克隆及生化特性。这些8-氧代鸟嘌呤DNA糖基化酶，hOgg1（人类）和mOgg1（小鼠），彼此同源且与酵母Ogg1同源。它们还含有一个活性位点基序——螺旋-发夹-螺旋、富含甘氨酸/脯氨酸-天冬氨酸基序——这是具有相似核心折叠和活性位点几何结构的BER蛋白超家族的特征。hOgg1和mOgg1对DNA中与OG相对的碱基均表现出极高的选择性，仅对与胞嘧啶碱基配对的OG高效起作用。此外，hOgg1和mOgg1无法处理包括8-氧代腺嘌呤在内的一系列其他损伤，但能与合成的无碱基位点类似物高亲和力结合。这些蛋白通过经典的糖基化酶/裂解酶催化机制起作用；催化必需的赖氨酸残基发生突变会导致催化效力丧失，但仍保留与含OG寡核苷酸的结合能力。hOGG1基因定位于3号染色体短臂（3p25/26）上，该区域在癌症中常发生缺失。
这些结果确凿地证实了人类和小鼠细胞中存在8-氧代鸟嘌呤DNA糖基化酶/裂解酶并确定了其特性，完善了共同保护哺乳动物免受鸟嘌呤氧化遗传毒性影响的三种蛋白质组合。观察到癌细胞中hOGG1至少一个等位基因常发生缺失，这表明此类细胞可能对抗活性氧诱变作用的能力降低，这种缺陷可能会增加其整体基因组不稳定性。最近的观察结果支持了这一推测，即持续激活Ras/Raf途径的细胞会持续产生高水平的超氧化物，而超氧化物是已知的OG生成剂。
