In Vitro Base Excision Repair Using Mammalian Cell Extracts

Base excision repair (BER) is an essential cellular mechanism that maintains genome stability by repairing DNA damage, such as DNA base lesions, base loss (AP sites) and single strand breaks, generated through endogenous metabolism or via exogenous mutagens. Therefore, in vitro BER assays are important for our understanding of the mechanism of cellular response to mutagens and may also reveal important information about the development of several DNA repair-related human diseases, such as cancer, and aging. Here, we describe the preparation and use of mammalian cell extracts in in vitro BER assays using both oligonucleotide and closed circular DNA substrates containing site-specific DNA lesions, in combination with denaturing acrylamide gel electrophoresis and phosphor imaging analysis.