Kamiya Hiroyuki, Yamazaki Daiki, Nakamura Eri, Makino Tetsuaki, Kobayashi Miwako, Matsuoka Ichiro, Harashima Hideyoshi
†Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
‡Graduate School of Science and Engineering, Ehime University, 2-5 Bunkyo-cho, Matsuyama 790-8577, Japan.
Chem Res Toxicol. 2015 Apr 20;28(4):621-8. doi: 10.1021/tx500418m. Epub 2015 Mar 2.
8-Oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA is one of the most important oxidatively damaged bases and causes G:C → T:A substitution mutations. The Werner syndrome protein (WRN) is a cancer-related RecQ DNA helicase and plays many roles in DNA replication and repair. To examine the relationships between G(O)-induced mutations and WRN, shuttle plasmid DNA containing a G(O):C pair in the supF gene was transfected into human U2OS cells, in which WRN was knocked down. The plasmid DNA replicated in the knockdown cells was introduced into an Escherichia coli indicator strain. The knockdown of WRN increased the mutant frequency of the G(O)-plasmid DNA. Unexpectedly, however, the WRN knockdown only slightly enhanced the targeted G:C → T:A mutation. Instead, base-substitution mutations at various positions were more frequently detected, with statistical significance. The results obtained in this study suggested that the reduction of the cancer-related WRN induced action-at-a-distance mutagenesis by the G(O):C pair in human cells. In addition, the WRN knockdown decreased the G(O):A-induced A:T → C:G mutations, suggesting that WRN may enhance the mutations caused by G(O) in the nucleotide pool.
DNA中的8-氧代-7,8-二氢鸟嘌呤（G(O)，8-羟基鸟嘌呤）是最重要的氧化损伤碱基之一，可导致G:C→T:A替换突变。沃纳综合征蛋白（WRN）是一种与癌症相关的RecQ DNA解旋酶，在DNA复制和修复中发挥多种作用。为了研究G(O)诱导的突变与WRN之间的关系，将在supF基因中含有G(O):C对的穿梭质粒DNA转染到WRN被敲低的人U2OS细胞中。在敲低细胞中复制的质粒DNA被导入大肠杆菌指示菌株。WRN的敲低增加了G(O) - 质粒DNA的突变频率。然而，出乎意料的是，WRN敲低仅略微增强了靶向的G:C→T:A突变。相反，在各个位置的碱基替换突变被更频繁地检测到，具有统计学意义。本研究获得的结果表明，癌症相关的WRN的减少在人细胞中通过G(O):C对诱导了远距离诱变。此外，WRN敲低降低了G(O):A诱导的A:T→C:G突变，表明WRN可能增强了核苷酸池中由G(O)引起的突变。
