IN-VITRO EUKARYOTIC DNA EXCISION-REPAIR ASSAYS - AN OVERVIEW

Great progress is being made in understanding the process of nucleotid e excision repair (NER) in eukaryotes. Different lines of research hav e been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage o f a cell-free system that can mediate DNA excision-repair by transcrip tionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the othe r undamaged as internal control. The extent of repair activity is dete rmined by following the level of radiolabeled incorporation during the repair synthesis step consecutive to the excision of DNA lesions. We discuss the interest and drawbacks of this biochemical assay in light of the main results obtained. We report the modifications that we have undertaken in order to determine repair synthesis activity in a chemi luminescent-directed reaction as well as to assess incision activity i n protein extracts.