Ohsaki Arisa, Iiyama Kazuhiro, Miyagawa Yoshitaka, Kawaguchi Yutaka, Koga Katsumi, Kusakabe Takahiro
Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Mol Biol Rep. 2005 Mar;32(1):25-34. doi: 10.1007/s11033-004-2474-y.
Nonhomologous end-joining (NHEJ) is one of the repair pathways for double-strand breaks (DSBs) in eukaryotic cells. By using linearized plasmid substrates, we have detected intramolecular NHEJ activity in a cell-free extract from the cultured silkworm cell line BmN4. The efficiency of NHEJ differed according to the structure of DNA ends; approximately 1% of input DNA was repaired when the substrate had cohesive ends. The reaction required the hydrolysis of nucleotide triphosphate; interestingly, all of four rNTPs or four dNTPs could support the reaction. A substrate with non-complementary DNA ends was mainly repaired by the DNA polymerase-mediated pathway. These results indicate that the present cell-free system will be useful to analyze the molecular mechanisms of DSB repair and NHEJ in insect cells.
非同源末端连接（NHEJ）是真核细胞中双链断裂（DSB）的修复途径之一。通过使用线性化质粒底物，我们在培养的家蚕细胞系BmN4的无细胞提取物中检测到了分子内NHEJ活性。NHEJ的效率因DNA末端的结构而异；当底物具有粘性末端时，约1%的输入DNA得到修复。该反应需要三磷酸核苷酸的水解；有趣的是，四种核糖核苷酸三磷酸（rNTPs）或四种脱氧核糖核苷酸三磷酸（dNTPs）都能支持该反应。具有非互补DNA末端的底物主要通过DNA聚合酶介导的途径进行修复。这些结果表明，目前的无细胞系统将有助于分析昆虫细胞中DSB修复和NHEJ的分子机制。
