Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells
Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells
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DOI：
10.1093/nar/24.10.1837
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发表时间：
1996-05-15
影响因子：
14.9
通讯作者：
Sarasin, A
中科院分区：
文献类型：
--
作者：
Gentil, A;LePage, F;Sarasin, A
利用穿梭载体在哺乳动物细胞中研究了紫外线诱导的光产物顺式胸腺嘧啶-胸腺嘧啶二聚体（TT）和胸腺嘧啶-胸腺嘧啶酮（6-4）光产物[T（6-4）T]的致突变特性。以单链DNA形式产生能够在哺乳动物细胞和细菌中复制的穿梭载体。在单链DNA载体的单个限制性位点处插入独特的光产物，并且在单链DNA载体的再环化之后，将后者转染到猿猴COS 7细胞中。扩增的DNA不经任何选择性筛选，直接测序。我们的结果清楚地表明，这两种损伤都是致突变的，但在不同的水平上，观察到TT二聚体和T（6-4）T的突变频率分别为2%和60%。对于TT二聚体，突变靶向3 '-T，对于T（6-4）T，观察到大量的各种突变。大多数G->T颠换半靶向光产物的5'-T之前的碱基。当相同的质粒直接转染到SOS诱导的JM 105细菌中或当插入不同质粒中的T（6-4）T寡核苷酸在SOS诱导的SMH 10大肠杆菌细菌中复制时，未观察到这些类型的突变。因此，这些半靶向突变是通过一种真核DNA聚合酶绕过COS 7细胞中的T（6-4）T病变的特异性结果。
The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form, A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells, After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced, Our results show clearly that both lesions are mutagenic, but at different levels, Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T, With the T(6-4)T a large,variety of mutations were observed, A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coli bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.