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 Polκ, 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 Polκ does not interfere with other polymerases repairing monoalkylation damage.