In vitro base excision repair assay using mammalian cell extracts

Base excision repair (BER) is the main pathway for removal of endogenous DNA damage. This repair mechanism is initiated by a specific DNA glycosylase that recognizes and removes the damaged base through N-glycosylic bond hydrolysis. The generated apurinic/apyrimidinic (AP) site can be repaired in mammalian cells by two alternative pathways which involve either the replacement of one (short patch BER) or more nucleotides (long patch BER) at the lesion site. This chapter describes a repair replication assay for measuring BER efficiency and mode in mammalian cell extracts. The DNA substrate used in the assay is either a randomly depurinated plasmid DNA or a plasmid containing a single lesion that is processed via BER (for example a single AP site or uracil residue). The construction of a single lesion at a defined site of the plasmid genome makes the substrate amenable to fine mapping of the repair patches, thus allowing discrimination between the two BER pathways.