Simultaneous Short- and Long-Patch Base Excision Repair (BER) Assay in Live Mammalian Cells.

Abstract

The base excision repair (BER) pathway repairs small, non-bulky DNA lesions, including oxidized, alkylated, and deaminated bases, and is responsible for the removal of at least 20,000 DNA lesions per cell per day. BER is initiated by DNA damage-specific DNA glycosylases that excise the damaged base and generates an abasic (AP) site or single-strand breaks, which are subsequently repaired in mammalian cells either by single-nucleotide (SN) or multiple-nucleotide incorporation via the SN-BER or long-patch BER (LP-BER) pathway, respectively. This chapter describes a plaque-based host cell reactivation (PL-HCR) assay system for measuring BER mechanisms in live mammalian cells using a plasmid-based assay. After transfection of a phagemid (M13mp18) containing a single modified base (representative BER DNA substrates) within a restriction site into human cells, restriction digestions detect the presence or absence (complete repair) of the adduct by the transformation of the digestion products into E. coli and counting the transformants as plaques. To monitor the patch size, different plasmids are constructed containing C:A mismatches within different restriction sites (inhibiting digestion) at various distances on both sides (5' or 3') of the modified base-containing restriction sites. Using this assay, the percentage of repair events that occur via 5' and 3' patch formation can be quantified.