Kamiya H, Iwai S, Kasai H
Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
Nucleic Acids Res. 1998 Jun 1;26(11):2611-7. doi: 10.1093/nar/26.11.2611.
Two major ultraviolet-induced photolesions of TpT, a (6-4) photoproduct [T(6-4)T] and a cis-syn cyclobutane TT dimer (T=T), were incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the modified DNAs were transfected into simian COS-7 cells. The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli. The DNA replication efficiencies of plasmids containing T(6-4)T and T=T in the template strand for lagging strand synthesis were 93 and 79%, respectively, as compared with the unmodified DNA. Similar inhibitory effects were observed in the cases of the photoproducts in the template strand for leading strand synthesis (71 and 58%, respectively). These results indicated that T(6-4)T blocked DNA replication more weakly than T=T during leading and lagging strand syntheses in mammalian cells. The mutation frequencies of T(6-4)T were 2.3 and 4.7% in the leading and lagging template strands, respectively. The T=T lesion was less mutagenic and induced mutations with 0.2-0.7% frequencies. The T(6-4)T lesion primarily elicited 3'-T-->C substitutions, and T=T induced various types of mutations. These results indicate that T(6-4)T is more mutagenic than T=T during leading and lagging strand syntheses in simian cells. Moreover, this is the first evidence that shows T(6-4)T mainly elicits targeted substitutions at its 3'-T site in mammalian cells.
在双链载体的前导链和后随链模板之一的预定位点引入了两种主要的紫外线诱导的TpT光损伤，即(6-4)光产物[T(6-4)T]和顺式-顺式环丁烷TT二聚体(T=T)，然后将修饰后的DNA转染到猴COS-7细胞中。回收细胞中复制的DNA，并再次转染到大肠杆菌中。与未修饰的DNA相比，后随链合成模板链中含有T(6-4)T和T=T的质粒的DNA复制效率分别为93%和79%。在前导链合成模板链中的光产物情况下也观察到类似的抑制作用(分别为71%和58%)。这些结果表明，在哺乳动物细胞的前导链和后随链合成过程中，T(6-4)T对DNA复制的阻断作用比T=T弱。T(6-4)T在前导链和后随链模板链中的突变频率分别为2.3%和4.7%。T=T损伤的致突变性较低，诱导突变的频率为0.2-0.7%。T(6-4)T损伤主要引发3'-T→C替代，而T=T诱导各种类型的突变。这些结果表明，在猴细胞的前导链和后随链合成过程中，T(6-4)T比T=T更具致突变性。此外，这是首次证明T(6-4)T在哺乳动物细胞中主要在其3'-T位点引发靶向替代的证据。
