Inhibition of kinase activity of phosphatase inactive mutants by 3′-phosphorylated DNA

<p><b>Copyright information:</b></p><p>Taken from "The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks"</p><p>Nucleic Acids Research 2006;34(8):2230-2237.</p><p>Published online 28 Apr 2006</p><p>PMCID:PMC1450335.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () hPNK mutant D171A readily phosphorylates substrates lacking a 3′-phosphate. The indicated oligonucleotide duplexes (50 nM) were incubated with hPNK D171A at the concentrations shown for 10 min at 37°C. After quenching, the samples were resolved in a 20% denaturing polyacrylamide gel and analysed by fluorimagery. () 3′-Phosphorylated oligonucleotide duplex inhibits processing of kinase substrate by D171A. Oligonucleotide duplex ‘Overhang’ (50 nM) was incubated with hPNK D171A (4 nM) for 8 min at 37°C in the presence of varying concentrations of 3′-phosphate-containing ‘Competitor’ duplex. After quenching, the samples were resolved in a 20% denaturing PAGE and analysed by fluorimagery. Data points represent the mean, and error bars the standard deviation of three independent experiments. Curve fitting was carried out by non-linear regression using SigmaPlot. The is calculated as the distance between the X-intercepts of the linear plots, which represent lines drawn from V, the highest point of the curve (i.e. in the absence of inhibitor), to cross the curve at V/2, V/3, V/4, V/5 as described by Dixon (). () Nitrocellulose filter-binding assay shows that single and double stranded 3′-phosphorylated substrates are efficiently bound by D171A. Binding reactions contained 100 pM FAM-labelled double-stranded (ds, ‘Overhang’, ) or single-stranded (ss, FAM1, ) substrate and concentrations of hPNK D171A ranging from 0.1–81 nM. The extent of binding was measured by nitrocellulose filter binding assay as detailed in the Materials and Methods section. Data points represent the mean, and error bars the standard deviation of three independent experiments. () Optimal inhibition is obtained with a recessed 3′-phosphate. Oligonucleotide duplex ‘Overhang’ (50 nM) was incubated with hPNK D171A (4 nM) for 8 min at 37°C in the presence of the indicated competitors (10 nM), where an asterisk corresponds to a 3′-phosphate. After quenching, the samples were resolved in a 20% denaturing PAGE and analysed by fluorimagery. Data points represent the mean, and error bars the standard deviation of three independent experiments