In vitro eukaryotic DNA excision repair assays: an overview.
Great progress is being made in understanding the process of nucleotide excision repair (NER) in eukaryotes. Different lines of research have been developed, among them an in vitro assay with cell-free extracts has played a major role. This in vitro repair assay takes advantage of a cell-free system that can mediate DNA excision-repair by transcriptionally active protein extracts from mammalian cells incubated in the presence of two plasmids of different sizes, one damaged and the other undamaged as internal control. The extent of repair activity is determined 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 chemiluminescent-directed reaction as well as to assess incision activity in protein extracts.
Salles
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B
Laboratoire de Pharmacologie et Toxicologie Fondamentales CNRS, Toulouse, France.
Frit
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P
Provot
C
C
Jaeg
J P
JP
Calsou
P
P
D000818:Animals; D004247:DNA / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D005057:Eukaryotic Cells / Q000502:physiology*; D005821:Genetic Techniques; D006801:Humans; D007553:Isotope Labeling; D008163:Luminescent Measurements
D004247:DNA