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.