Quantitative analysis of Werner helicase activity using the single-molecule fluorescence detection system MF10S.
We developed a system that uses the single-molecule fluorescence detection system MF10S to assess quantitatively the activity of WRN helicase, the product of the causative gene of Werner syndrome that includes premature ageing. Double-strand DNA substrates labeled with the fluorescence dye TAMRA at the 5' end and with a quencher at the 3' end of the counter strand were incubated with a single trapper oligonucleotide and Werner helicase, and the resultant single DNA fragments labeled with TAMRA produced by the unwinding of WRN helicase were detected using the MF10S. The results using this system and those using polyacrylamide gel electrophoresis were well correlated. The MF10S system provides a quantitative analysis that is much faster, simpler, and more economical than systems using polyacrylamide gel electrophoresis and radioisotopes, and could be used as a quantitative analysis system for Werner helicase and other DNA helicase activities.
Futami
Kazunobu
K
GeneCare (previously AGENE) Research Institute, Kamakura, Kanagawa, Japan.
Goto
Hideyuki
H
Shimamoto
Akira
A
Watanabe
Takahide
T
Furuichi
Yasuhiro
Y
D004265:DNA Helicases / Q000032:analysis* / Q000378:metabolism; D005090:Exodeoxyribonucleases; D006801:Humans; D053484:RecQ Helicases; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000592:standards; D014898:Werner Syndrome / Q000201:enzymology* / Q000235:genetics; D000071657:Werner Syndrome Helicase
D005090:Exodeoxyribonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase