Kamiya Hiroyuki, Makino Tetsuaki, Suzuki Tetsuya, Kobayashi Miwako, Matsuoka Ichiro
Graduate School of Biomedical and Health Sciences, Hiroshima University, Minami-ku, Hiroshima, Japan.
Graduate School of Science and Engineering, Ehime University, Matsuyama, Japan.
Mutagenesis. 2018 Oct 11;33(4):301-310. doi: 10.1093/mutage/gey024.
Reactive oxygen species generate 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine), which induces G:C→T:A transversion mutations. The knockdowns of the protein responsible for Werner syndrome (WRN), a cancer-associated DNA helicase, and DNA polymerase (pol) λ, a WRN-interacting DNA pol, cause untargeted base-substitution mutations (action-at-a-distance mutations). To examine the consequences of the dual reductions of WRN and pol λ for the mutations caused by GO, siRNAs against both proteins were introduced into human U2OS cells. A replicable plasmid DNA with the oxidised nucleobase in a unique position of the supF gene was then introduced into the double knockdown cells. The amplified DNA recovered from the cells was used to transform a bacterial indicator strain. The mutant frequency and the subsequent sequence analysis revealed that the double knockdown additively promoted the G:C→T:A substitution at the GO position and increased the action-at-a-distance mutations to a level similar to that of the single WRN knockdown. Thus, WRN and DNA pol λ seem to suppress the targeted G:C→T:A mutation at least in part independently and reduce the untargeted mutations via an identical pathway.
活性氧会生成8-氧代-7,8-二氢鸟嘌呤（GO，8-羟基鸟嘌呤），其会诱导G:C→T:A颠换突变。与癌症相关的DNA解旋酶——沃纳综合征（WRN）相关蛋白以及与WRN相互作用的DNA聚合酶（pol）λ的敲低会导致非靶向碱基取代突变（远距离作用突变）。为了研究WRN和pol λ双重减少对GO引起的突变的影响，针对这两种蛋白的小干扰RNA（siRNA）被导入人U2OS细胞。然后，将在supF基因独特位置带有氧化核碱基的可复制质粒DNA导入双重敲低细胞。从细胞中回收的扩增DNA用于转化细菌指示菌株。突变频率及后续的序列分析表明，双重敲低会累加促进GO位置的G:C→T:A替换，并将远距离作用突变增加到与单一WRN敲低相似的水平。因此，WRN和DNA pol λ似乎至少部分独立地抑制靶向的G:C→T:A突变，并通过相同途径减少非靶向突变。
