Gentil A, Le Page F, Cadet J, Sarasin A
Laboratoire de Génétique Moléculaire, Villejuif, France.
Mutat Res. 2000 Jul 20;452(1):51-6. doi: 10.1016/s0027-5107(00)00034-8.
Ionizing radiations often induce multiple and clustered DNA lesions at the site of DNA interaction. As a model, we have studied the toxicity and the mutagenicity of two adjacent oxidative bases as clustered DNA lesions in mammalian cells using shuttle vectors. The chosen oxidative lesions were 8-oxo-7,8-dihydroguanine, the formylamine residue resulting from the oxidation of a pyrimidine base and the tandem lesion 8-oxo-7,8-dihydroguanine/formylamine where both modifications are located at a vicinal position. A single-stranded DNA shuttle vector carrying a unique DNA lesion was constructed, transfected into simian COS7 cells and mutations induced after replication in mammalian cells were screened in bacteria. 8-oxo-7,8-dihydroguanine, as expected, does not affect greatly survival (70% bypass) whereas formylamine and the tandem lesions are blocking alterations, DNA polymerase bypass being of 45% and 17%, respectively. Base insertion opposite the lesion was studied. Under our experimental conditions, replication of 8-oxo-7, 8-dihydroguanine finally gives rise to guanine:cytosine pairing, rendering this lesion only slightly mutagenic. This is not the case for the formylamine that codes preferentially for adenine (71%). In addition, one-base deletions were observed targeted to the site to the lesion. Cytosine and thymine were inserted opposite the lesion with similar but low frequencies. Thus, coding properties of the formylamine render this residue very mutagenic when coming from the oxidative alteration of a cytosine. The coding properties of the tandem damage are a combination of the contribution of the two isolated lesions with a very high percentage of adenine insertion (94%) opposite the formylamine residue of the tandem lesion. The toxicity as well as the mutation spectrum of the tandem lesion allow us to speculate about the molecular mechanism with which the DNA polymerase replicates these two lesions.
电离辐射常常在DNA相互作用位点诱导产生多个成簇的DNA损伤。作为一个模型，我们使用穿梭载体研究了作为哺乳动物细胞中成簇DNA损伤的两个相邻氧化碱基的毒性和致突变性。所选择的氧化损伤是8-氧代-7,8-二氢鸟嘌呤、嘧啶碱基氧化产生的甲酰胺残基以及两个修饰均位于相邻位置的串联损伤8-氧代-7,8-二氢鸟嘌呤/甲酰胺。构建了携带单个DNA损伤的单链DNA穿梭载体，将其转染到猴COS7细胞中，并在细菌中筛选哺乳动物细胞复制后诱导产生的突变。如预期的那样，8-氧代-7,8-二氢鸟嘌呤对细胞存活影响不大（70%的损伤可被绕过），而甲酰胺和串联损伤则是阻断性改变，DNA聚合酶绕过损伤的比例分别为45%和17%。研究了损伤对面的碱基插入情况。在我们的实验条件下，8-氧代-7,8-二氢鸟嘌呤的复制最终导致鸟嘌呤:胞嘧啶配对，使得该损伤仅具有轻微的致突变性。甲酰胺的情况则不同，它优先编码腺嘌呤（71%）。此外，观察到损伤位点存在靶向的单碱基缺失。胞嘧啶和胸腺嘧啶以相似但较低的频率插入到损伤对面。因此，当甲酰胺来自胞嘧啶的氧化改变时，其编码特性使其具有很强的致突变性。串联损伤的编码特性是两个孤立损伤作用的组合，串联损伤中甲酰胺残基对面的腺嘌呤插入比例非常高（94%）。串联损伤的毒性以及突变谱使我们能够推测DNA聚合酶复制这两个损伤的分子机制。
