Base Excision Repair in Mammalian Cells

A rapid, convenient and safe in vitro assay system for base excision repair is described. Whole cell extracts are prepared by detergent-based cell lysis and provide a vigorous activity of AP site repair. A circular DNA substrate is used for detection of both DNA polymerase β-dependent and proliferating cell nuclear antigen (PCNA)-dependent pathways. Repaired and unrepaired DNA substrates are separated by agarose gel electrophoresis as a linear DNA molecule and a nicked circular molecule, respectively, and detected by staining with SYBR Green I. This assay system does not require radioactive substrates or nucleotides, and provides a sensitivity in which 10 ng of a DNA substrate per reaction is sufficient for quantitative repair analysis.