Shimizu H, Yagi R, Kimura Y, Makino K, Terato H, Ohyama Y, Ide H
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan.
Nucleic Acids Res. 1997 Feb 1;25(3):597-603. doi: 10.1093/nar/25.3.597.
alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for alpha were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site. The transfection assay revealed that alpha constituted a moderate block to DNA replication. The in vivo replication capacity to pass through alpha was approximately 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block. Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E.coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of alpha introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G. A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model.
α-2'-脱氧腺苷（α）是在缺氧条件下γ射线辐照DNA产生的主要腺嘌呤损伤。在本研究中，构建了含有α的单链重组M13载体，并将其转染到大肠杆菌中，以评估该损伤的致死和诱变效应。将α的数据与在相同位点含有正常A或模型无碱基位点（F）的M13载体获得的数据进一步进行比较。转染试验表明，α对DNA复制构成中度阻碍。相对于正常A，通过α的体内复制能力约为20%，但比构成几乎绝对复制阻碍的F高20倍。通过大肠杆菌DNA聚合酶I对含有α或F的寡核苷酸模板进行体外复制也获得了类似的数据。通过对后代噬菌体进行直接DNA测序，分析了复制含有α的M13 DNA的诱变后果。诱变完全针对引入载体中的α位点。突变仅为单个核苷酸缺失，未检测到碱基替换。基于错配插入-链滑动模型讨论了与α相关的单个核苷酸缺失的可能机制。
