Gasparutto Didier, Dhérin Claudine, Boiteux Serge, Cadet Jean
Laboratoire des Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR 5046 CEA-CNRS-UJF, Département de Recherche Fondamentale sur la Matière Condensée, CEA-Grenoble, 17 Avenue des Martyrs F-38054 Cedex 9, Grenoble, France.
DNA Repair (Amst). 2002 Jun 21;1(6):437-47. doi: 10.1016/s1568-7864(02)00016-2.
8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.
8-甲基-2'-脱氧鸟苷（8-medGuo）已被证明是甲基自由基攻击DNA诱导的2'-脱氧鸟苷的主要稳定烷基化产物。此外，通过引物延伸分析，最近有报道称，后一种DNA修饰通过在体外产生G到C和G到T的颠换及缺失而成为一种错义损伤。然而，到目前为止，尚未有关于DNA修复机制对这种C8-烷基化核苷进行处理的数据报道。因此，我们研究了几种细菌、酵母和哺乳动物DNA N-糖基化酶对特异性掺入寡核苷酸底物中的8-甲基鸟嘌呤（8-meGua）位点的切除能力。结果表明，来自大肠杆菌的3-甲基腺嘌呤（3-meAde）DNA糖基化酶II（AlkA蛋白）是唯一测试过的能够从双链DNA片段中去除8-meGua的DNA N-糖基化酶。此外，AlkA对8-meGua的活性根据DNA中的互补碱基而有显著差异，与腺嘌呤和胸腺嘧啶配对时活性最高，与胞嘧啶和鸟嘌呤配对时活性最低。将AlkA蛋白对8-meGua的去除与7-甲基鸟嘌呤（7-meGua）和次黄嘌呤（Hx）的去除进行了比较。作为AlkA底物的损伤程度排序为7-meGua>8-meGua>Hx。相比之下，人类3-meAde DNA N-糖基化酶（Mpg）无法释放与四种DNA碱基中任何一种配对的8-meGua。我们还表明，参与去除氧化损伤的DNA N-糖基化酶，如大肠杆菌的Fpg或Nth蛋白、酿酒酵母的Ntg1、Ntg2或Ogg1蛋白，或人类Ogg1，都不能释放与四种DNA碱基中任何一种配对的8-meGua。此外，HeLa细胞和中国仓鼠卵巢（CHO）细胞的无细胞蛋白提取物对与腺嘌呤或胞嘧啶配对的8-meGua没有任何切割活性，这表明哺乳动物细胞中不存在对这种损伤的碱基切除修复（BER）。
