Nonhomologous end-joining in a cell-free extract from the cultured silkworm cell line BmN4.
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.
Ohsaki
Arisa
A
Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
Iiyama
Kazuhiro
K
Miyagawa
Yoshitaka
Y
Kawaguchi
Yutaka
Y
Koga
Katsumi
K
Kusakabe
Takahiro
T
D000818:Animals; D012831:Bombyx / Q000737:chemistry / Q000166:cytology / Q000235:genetics*; D002413:Cations, Divalent / Q000737:chemistry; D002460:Cell Line; D002474:Cell-Free System; D004249:DNA Damage; D004260:DNA Repair; D008670:Metals / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic
D002413:Cations, Divalent; D008670:Metals