Introduction and characterization of a polymerase-dead point mutation into the POLK gene in vertebrates.
The chicken DT40 cell line is widely used for gene knock-outs. We attempted to introduce a polymerase-dead point mutation into Polkappa, a polymerase for translesion DNA synthesis, taking advantage of the highly efficient targeted integration in DT40 cells. The resulting cells (REV3(-/-)POLK(/)(pol-dead)) proliferated with the same kinetics as the parental REV3(-/-) cells. Though the mock-treated REV3(-/-)POLK(/)(mock) cells showed the same sensitivity as the parental REV3(-/-) cells to methyl methanesulfonate, the REV3(-/-)POLK(/)(pol-dead) cells demonstrated the same sensitivity as the REV3(-/-)POLK(/-) double knock-out cells. This implies that the presence of the polymerase-dead Polkappa does not interfere with other polymerases repairing monoalkylation damage.
Takenaka
Katsuya
K
Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo, 113-8510 Tokyo, Japan.
Miki
Yoshio
Y
D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D002645:Chickens; D002999:Clone Cells; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D055786:Gene Knockout Techniques; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity; D017354:Point Mutation; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D014162:Transfection; D014714:Vertebrates / Q000235:genetics* / Q000378:metabolism
D009153:Mutagens; D001216:Asparagine; D008741:Methyl Methanesulfonate; D004259:DNA-Directed DNA Polymerase