A PCR Based Method to Quantify DNA Excision Repair

Helix-distorting DNA base damages halt the activity of DNA and RNA polymerases and can lead to stress, mutations, or cell death. These include pyrimidine dimers induced by UV radiation, Benzo[a]pyrene diol epoxide (BPDE) adducts induced by smoking, and adducts induced by the chemotherapy cisplatin. The major mechanism for the repair of these damages in human cells is nucleotide excision repair (NER). To date, measuring NER efficiency in cells required specialized reagents and expertise usually available only in DNA repair labs. Here we describe a simple method to measure helix-distorting DNA damages and their repair – that can be implemented in any lab with basic molecular biology capacities. This method is based on long-amplicon PCR (LA-PCR) using a DNA polymerase which is blocked by damaged DNA. Damaging cells results in lower LA-PCR signal in comparison to non-damaged DNA, and repair is measured as restoration of PCR signal over time. We show that the method efficiently detects UV, BPDE and cisplatin induced damages and repair. As expected, repair measured by LA-PCR in heterochromatic regions is less efficient than repair in transcribed and accessible regions. Furthermore, lower repair is measured in NER-deficient knock-out cells. Together, our results indicate the method could be applied by non-DNA repair experts to study the involvement of their gene-of-interest in repair or the repair of their locus-of-interest in the genome. Furthermore, this method could be used to screen for novel factors involved in NER in specific genomic loci.