Host: The Japan Radiation Research Society
Base excision repair (BER), a predominant pathway to repair small base damages, includes two sub-pathways, short-patch which is dependent on polymerase β (polβ) and long-patch BER which is dependent on PCNA. Recent studies have shown that mouse polβ knockout cells cannot repair the methylated bases by BER in both G1 and S phases. But it is possible that other BER enzymes can be affected in the absence of polβ. In this study, to compare the mode of BER in human cells, we generated a stable human cell line in which polβ expression has been decreased using RNA interference and analyzed the effect of methyl methansulfonate (MMS) in wild type and knockdown cells. We first found the target RNA sequence and constructed the plasmid which expressed shRNA. After transfection to HeLa RC355 cells, we obtained the stable zeocin resistant cells and analyzed siRNA-induced 50% silencing of polβ using Western Blot. The cells were treated for 1h with different concentrations of MMS and examined the survival after 10 days. The number of colonies of knockdown cells was apparently decreased at 1.5mM or higher MMS concentrations. These results suggest the vital role of human polβ in the repair of MMS-induced damages.
