A new flow cytometric method to follow DNA gap filling during nucleotide excision repair of UVc-induced damage.
BACKGROUND
Several methods have been developed for studying the kinetics of DNA repair after exposure of cells to ultraviolet (UV) light, such as conventional assays measuring unscheduled DNA synthesis (UDS). In this study, we have developed an accurate and rapid method to follow DNA gap filling during nucleotide excision repair (NER) in normal human fibroblasts (NHFs) in response to UV-induced damage.


METHODS
After UVc irradiation, aphidicolin was added to the culture to hold repair patches open. This allowed an efficient incorporation of biotin-21-dUTP during an endogenous DNA repair synthesis that was detected by flow cytometry.


RESULTS
We showed that the DNA gap filling after UVc irradiation in NHFs increased with time up to 10 h after irradiation and that no repair synthesis activity could be detected in XP-A fibroblasts. Furthermore, this activity was UVc dose dependent up to 20 J/m2. These results correlated well with those of the UDS assay. Interestingly, addition of aphidicolin at different time points after UVc irradiation, thus allowing endogenous repair synthesis in the absence of biotin-21-dUTP, demonstrated that the response of the NER system occurred extremely rapidly after irradiation.


CONCLUSIONS
This method may be a reliable and simple alternative to other techniques measuring UDS. Practical advantages include the rapidity of the method, no need for radioactivity, and the possibility to use a second and/even a third flow marker to analyse cell cycle and heterogeneous cell populations concomitantly.
Vincent
F
F
Laboratoire de Cancérologie Expérimentale et de Radiobiologie, Institut de Recherche contre les Cancers de l'Appareil Digestif, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. Florence.Vincent@ircad.u-strasg.fr
Ceraline
J
J
Goldblum
S
S
Klein-Soyer
C
C
Bergerat
J P
JP
D016590:Aphidicolin / Q000494:pharmacology; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D013997:Time Factors; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism
D004791:Enzyme Inhibitors; D016590:Aphidicolin