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 Pol kappa, 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 Pol kappa does not interfere with other polymerases repairing monoalkylation damage. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.