A REPAIR COMPETITION ASSAY TO ASSESS RECOGNITION BY HUMAN NUCLEOTIDE EXCISION-REPAIR

We developed a competition assay to compare, in a quantitative manner, the ability of human nucleotide excision repair (NER) to recognise st ructurally different forms of DNA damage. This assay uses a NER substr ate consisting of M13 double-stranded DNA with a single and uniquely l ocated acetylaminofluorene (AAF) adduct, and measures the efficiency b y which multiply damaged plasmid DNA competes for excision repair of t he site-directed modification, To validate this assay, we tested compe titor DNA containing defined numbers of either AAF adducts or UV radia tion products, In both cases, repair of the site-directed NER substrat e was inhibited in a damage-specific and dose-dependent manner. We the n exploited this competition assay to determine the susceptibility of bulky adozelesin-DNA adducts to human NER.