Vidal A E, Hickson I D, Boiteux S, Radicella J P
Département de Radiobiologie et Radiopathologie, Commissariat à l'Energie Atomique, UMR217 CNRS-CEA, BP6, F92265 Fontenay aux Roses, France.
Nucleic Acids Res. 2001 Mar 15;29(6):1285-92. doi: 10.1093/nar/29.6.1285.
The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.
细胞中活性氧的产生会引发多种损伤，其中包括DNA中8-氧代-7,8-二氢鸟嘌呤（8-氧代鸟嘌呤，8-oxoG）的形成。由于在复制过程中与腺嘌呤错配，8-氧代鸟嘌呤具有高度致突变性。为了将这种氧化嘌呤的致突变潜力降至最低，人类细胞具有一种特异性的8-氧代鸟嘌呤DNA糖基化酶/AP裂解酶（hOGG1），它启动8-氧代鸟嘌呤的碱基切除修复（BER）。我们在此表明，在体外，BER途径的这第一种酶效率相对较低，因为它对其催化反应的产物具有高亲和力（复合物的半衰期>2小时），导致hOGG1周转不足。然而，hOGG1的糖基化酶活性受到主要的人类AP核酸内切酶HAP1（APE1）的刺激，HAP1是在BER中执行后续步骤的酶，同时也受到催化无活性的突变体（HAP1-D210N）的刺激。在HAP1存在的情况下，hOGG1 DNA糖基化酶产生的AP位点可被核酸内切酶占据，从而避免hOGG1重新结合。此外，糖基化酶对未切割的AP位点的亲和力高于对HAP1产生的切割后DNA产物的亲和力。这将使平衡向游离糖基化酶方向移动，使其能够启动新的催化循环。相反，HAP1不影响hOGG1的AP裂解酶活性。仅对hOGG1糖基化酶反应的这种刺激加剧了其糖基化酶和AP裂解酶活性的解偶联。这些数据表明，在HAP1存在的情况下，绕过hOGG1的AP裂解酶活性并因此排除一个潜在的限速步骤，8-氧代鸟嘌呤残基的BER可以非常高效。
