Repair Synthesis Assay for Nucleotide Excision Repair Activity Using Fractionated Cell Extracts and UV-Damaged Plasmid DNA

Methods are described for measuring nucleotide excision repair (NER) of damaged plasmid DNA using fractionated mammalian cell extracts. NER creates a single-stranded gap of approx 25–30 nt. Filling of this gap by repair synthesis can be monitored by the incorporation of radioactive nucleotides. We first describe the preparation of ultraviolet light (UV)-damaged and control plasmid DNA substrates and purification of their closed-circular forms. To increase the specificity for NER, plasmid molecules containing pyrimidine hydrates and other lesions sensitive to Escherichia coli Nth protein are eliminated. The preparation of whole cell extracts active in NER is described, both for cells grown as attached cultures and those grown in suspension. Cell extracts are partially purified on phosphocellulose to produce a fraction that can carry out the full NER reaction when combined with purified RPA and PCNA proteins. This enables NER to be quantified in an assay with exceptionally low background in nondamaged DNA.