Equilibrium binding of single strand DNA (P1) from promoter with mNdK

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 CD of folded P1 and decay in intrinsic mNdK fluorescence owing to P1 binding were used to characterize the P1-mNdK interaction. () mNdK binding disrupts folded P1 motif. CD spectra of P1 (4 μM) shows decrease in ellipticity at 285 nm with increasing mNdK (0–0.5 μM) in 50 mM Tris-MES-acetate pH 6.5, 1 mM MgCl, 75 mM NaCl within 30 min of incubation, after each addition, at 37°C. () Enzymatic (phosphotransfer) activity of mNdK at pH 6.5. An aliquot of 10 μCi of [γ-P]ATP was incubated with either no protein (lane 1) or 1 μM of mNdK at the indicated pH along with 1 mM unlabeled GDP. Experiments were done in 20 μl of 50 mM Tris–HCl, 5 mM MgCl, 1 mM DTT, pH 7.4 (lanes 1 and 2) or 50 mM Tris–MES–acetate, 5 mM MgCl, 1 mM DTT, pH 6.5 (lane 3) for 3 min at room temperature. Reactions were stopped with 2 μl 10× SDS buffer and separated using thin layer chromatography (PEI-TLC) in 0.75 M KHPO to resolve ATP and GTP. Analysis was done by autoradiography. () Fluorimetric determination of the equilibrium binding constant for P1 binding to mNdk. mNdK (0.5 μM) was titrated with P1 (20–420 nM) and the intrinsic protein fluorescence owing to tryptophan was monitored in the 300–420 nm range. () The change in fluorescence at 340 nm was plotted as a function of total P1 concentration. Inset: The fractional saturation, , was plotted versus [P1]/ according to , as described in Materials and Methods. The determined by this method was 65.1 ± 6.6 nM. Fluorescence decay owing to photobleaching within the same time was ∼6%; observed separately without P1. Experiments were done in 50 mM Tris-Cl, pH 7.4 containing 1 mM MgCl and 75 mM NaCl at 37°C.