Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity.
Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.
Collini
Maddalena
M
Department of Physics, University of Milano-Bicocca, Piazza della Scienza 3, 20126 Milan, Italy.
Caccia
Michele
M
Chirico
Giuseppe
G
Barone
Flavia
F
Dogliotti
Eugenia
E
Mazzei
Filomena
F
D045647:DNA Glycosylases / Q000378:metabolism*; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D017785:Photons; D013050:Spectrometry, Fluorescence / Q000379:methods*; D051981:Uracil-DNA Glycosidase
D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase