Girard P M, D'Ham C, Cadet J, Boiteux S
Département de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA, Radiobiologie Moléculaire et Cellulaire, Fontenay aux Roses, France.
Carcinogenesis. 1998 Jul;19(7):1299-305. doi: 10.1093/carcin/19.7.1299.
The yOgg1 protein of Saccharomyces cerevisiae is a DNA glycosylase/AP lyase that excises guanine lesions such as 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy-G) and incises apurinic/apyrimidinic sites (AP sites) in damaged DNA. The yOgg1 protein displays a marked preference for DNA duplexes containing 8-OxoG or AP sites placed opposite cytosine. In this paper, we show that yOgg1 can also excise an adenine lesion, 7,8-dihydro-8-oxoadenine (8-OxoA), when paired with cytosine or 5-methylcytosine. In contrast, yOgg1 does not release 8-OxoA when placed opposite thymine, adenine, guanine or uracil. The specificity constants (Kcat/Km) for repair of 8-OxoG/C and 8-OxoA/C duplexes are (50 +/- 18) x 10(-3) and (13 +/- 3) x 10(-3)/min/nM, respectively. The catalytic mechanism for strand cleavage at 8-OxoA/C involves excision of 8-OxoA by the DNA glycosylase activity of yOgg1, followed by incision at the newly formed AP site via a beta-elimination reaction. Furthermore, cleavage of 8-OxoA/C involves formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydride (NaBH4). The yOgg1 protein binds strongly to the 8-OxoA/C duplex, as demonstrated by an apparent dissociation constant (Kdapp) value of 45 nM, as determined by gel mobility shift assay. In contrast, the yOgg1 protein has a very low binding affinity for the 8-OxoA/T duplex, a Kdapp value of 680 nM, which in turn can explain the lack of repair of 8-OxoA in this duplex. The capacity of other DNA glycosylases/AP lyases to repair 8-OxoA has also been investigated. The results show that human hOgg1 protein efficiently repairs 8-OxoA placed opposite cytosine or 5-methylcytosine. On the other hand, the Fpg protein of Escherichia coli cleaves 8-OxoA/C at a very slow rate as compared with yOgg1.
酿酒酵母的yOgg1蛋白是一种DNA糖基化酶/AP裂解酶，可切除鸟嘌呤损伤，如7,8-二氢-8-氧代鸟嘌呤（8-氧代鸟嘌呤）和2,6-二氨基-4-羟基-5-N-甲基甲酰胺基嘧啶（甲基-Fapy-G），并在受损DNA中切割无嘌呤/无嘧啶位点（AP位点）。yOgg1蛋白对含有与胞嘧啶相对的8-氧代鸟嘌呤或AP位点的DNA双链体表现出明显的偏好。在本文中，我们表明，当与胞嘧啶或5-甲基胞嘧啶配对时，yOgg1也可以切除腺嘌呤损伤7,8-二氢-8-氧代腺嘌呤（8-氧代腺嘌呤）。相反，当8-氧代腺嘌呤与胸腺嘧啶、腺嘌呤、鸟嘌呤或尿嘧啶相对时，yOgg1不会释放它。修复8-氧代鸟嘌呤/胞嘧啶和8-氧代腺嘌呤/胞嘧啶双链体的特异性常数（Kcat/Km）分别为（50±18）×10^(-3)和（13±3）×10^(-3)/分钟/纳摩尔。8-氧代腺嘌呤/胞嘧啶处链切割的催化机制包括通过yOgg1的DNA糖基化酶活性切除8-氧代腺嘌呤，然后通过β-消除反应在新形成的AP位点进行切割。此外，8-氧代腺嘌呤/胞嘧啶的切割涉及形成一种反应中间体，该中间体在硼氢化钠（NaBH4）存在下转化为稳定的共价加合物。凝胶迁移率变动分析表明，yOgg1蛋白与8-氧代腺嘌呤/胞嘧啶双链体紧密结合，表观解离常数（Kdapp）值为45纳摩尔。相反，yOgg1蛋白对8-氧代腺嘌呤/胸腺嘧啶双链体的结合亲和力非常低，Kdapp值为680纳摩尔，这反过来可以解释该双链体中8-氧代腺嘌呤缺乏修复的原因。还研究了其他DNA糖基化酶/AP裂解酶修复8-氧代腺嘌呤的能力。结果表明，人类hOgg1蛋白能有效修复与胞嘧啶或5-甲基胞嘧啶相对的8-氧代腺嘌呤。另一方面，与yOgg1相比，大肠杆菌的Fpg蛋白切割8-氧代腺嘌呤/胞嘧啶的速度非常慢。
