FRET analysis of mNdK-catalyzed cleavage of single strand DNA (FP1T)

Copyright information:Taken from "Nucleoside diphosphate kinase from cleaves single strand DNA within the human promoter in an enzyme-catalyzed reaction"Nucleic Acids Research 2005;33(8):2707-2714.Published online 11 May 2005PMCID:PMC1097768.© The Author 2005. Published by Oxford University Press. All rights reserved Decay in FRET of the 5′-fluorescein and 3′-TAMRA labeled pyrimidine-rich single strand from the promoter in the presence of mNdK was analyzed as described in Materials and Methods. () FRET of the molecular beacon FP1T with mNdK. Spectrum (0 h) showing energy transfer in FP1T emission at 575 nm (acceptor TAMRA) is owing to the energy transfer from donor (fluorescein). Arrow indicates decrease in 575 nm after the specified times (1 and 18 h) following addition of mNdK (0.25 nM) to P1 (40 nM). Grey line (PK) shows spectra of FP1T on treatment with proteinase K (200 μg/ml) after 18 h of incubation with mNdK. Inset: initial (3 min) rate of decay at 575 nm with varying concentration of FP1T (20–400 nM) and 0.25 nM mNdK. () Lineweaver–Burk plots of the cleavage reaction of FP1T (20–400 nM) by mNdK (0.25 nM) with 0 mM (squares), 0.5 mM (circles) or 1 mM (triangles) of added GDP. FRET was observed, in all cases, on excitation at 480 nm. All experiments were done in 50 mM Tris–HCl, pH 7.4 containing 1 mM MgCl and 75 mM NaCl at 37°C and values are averaged from at least three independent experiments.