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.