Comet assay detects cold repair of UV-A damages in a human B-lymphoblast cell line.
During DNA repair studies, cells are occasionally kept on ice in order to suppress DNA repair. In the present studies cultivated human NC37 B-lymphoblasts were damaged by UV-A irradiation (365 nm) and DNA single strand breaks were detected at the single cell level with the alkaline comet assay in the temperature range from 4 degrees C to 44 degrees C. Single cell studies, in contrast to bulk experiments, allow to identify apoptotic or necrotic cells, which can be omitted for data analysis. Unexpectedly, similarly efficient single phase repair kinetics was found at all temperatures below 37 degrees C, i.e., particularly also in the cold. For recovery times below 20 min a linear decrease of DNA damage was detected. After 20 min, no additional repair was observed, i.e., complete repair of single strand breaks was not achieved. At 44 degrees C DNA damage increased with time, probably due to heat damage and cell death. Nucleotide excision repair inhibitors such as aphidicolin, 1-beta-D-arabinofuranosyl cytosine (araC) and hydroxyurea, but not the base excision repair inhibitor methoxyamine caused a strong increase in DNA strand breaks. The use of repair inhibitors confirmed DNA repair at 4 degrees C. In conclusion, partial repair of UV-A damage is similar at 37 degrees C and 4 degrees C and is probably governed by nucleotide excision repair. Keeping samples on ice may not result in a total suppression of DNA repair.
Bock
C
C
Institut für Molekulare Biotechnologie, Jena, Germany.
Dittmar
H
H
Gemeinhardt
H
H
Bauer
E
E
Greulich
K O
KO
D001402:B-Lymphocytes / Q000502:physiology / Q000528:radiation effects; D002460:Cell Line; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D006801:Humans; D013696:Temperature; D014466:Ultraviolet Rays
D004247:DNA