MNdK-induced cleavage of P1 and inhibition of cleavage by ATP

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 All reactions had 10 nM P1 (or dT31), 5′-end labeled with [γ-P]ATP. Reactions were performed in 50 mM Tris–HCl, pH 7.4, 1 mM (or 20 mM) MgCl and 75 mM NaCl for 10 min at 37°C in the presence of 1 μM single strand poly(dA) as non-specific competitor. () Autoradiography after separation of cleaved DNA on mNdK treatment. Lane 1,10 nM dT31 in presence of 2 mM mNdK; lanes 2–7, P1 with 0, 10, 50, 100, 500 or 2000 nM mNdk. () The extent of cleavage in () is represented as percentage of cleaved P1 versus concentration of mNdK. Amount of cleaved P1 was estimated from image quantification of the uncleaved band. () P1 cleavage with mNdK mutants. Lane 1, P1 only; lanes 2 and 3, P1 with 1 and 2 μM K10A; lanes 3 and 4, P1 with 1 and 2 μM H117A. () ATP inhibits mNdK-induced cleavage of P1, which is not abrogated at high Mg concentration. All reactions contained P1 and 0.2 μM mNdK. Lane 1, 0 mM ATP; lane 2, 2 mM ATP; lane 3, 2 mM ATP in the presence of 20 mM Mg. Cleaved products were separated in a 8% native polyacrylamide gel by electrophoresis in 0.5× TBE buffer at room temperature. Gels were vacuum dried and analyzed using Phosphorimager; image quantification was done using IMAGE QUANT software. Quantification of cleaved product was done from at least three independent experiments.