6612 Nucleic Acids Research, 2012, Vol. 40, No. 14 Figure 2. DNA 30-phosphatase activity of recombinant PNKP harbouring mutations associated with MCSZ. (A and B) Aliquots of the recombinant WT and mutant human PNKP (0.7–1 mg) employed in these experiments was fractionated by SDS–PAGE and visualized by staining with Coomassie brilliant blue. Note that the PNKP proteins depicted in panel A were puriﬁed by IMAC/cation exchange and employed in panel C, below, whereas those depicted in panel B were puriﬁed by IMAC alone and employed in panel D, below. (C and D) DNA 30-phosphatase activities of WT and mutant PNKP. 25 nM of the indicated oligonucleotide duplex substrate (top) was incubated without ( ) or with 2.5, 10, 25 or 50 nM of WT or the indicated mutant PNKP protein for 60 min at 30 C. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. A representative gel is shown on the left, and quantiﬁcation from four independent experiments (±SEM) on the right. For quantiﬁcation, the signal intensities of the 17-P substrate and 17-OH product bands were quantiﬁed using Aida Image Analyser v.427 and the percentage of 17-OH product calculated. The oligonucleotide duplex contains a 1-bp gap with 30-phosphate and 50-phosphate termini. The position of the 30-phosphate (circled) and 32P-label on the 17-mer substrate-strand are indicated (top). extract from MC5403 LCLs, suggesting that the greater residual level of PNKPE326K in these extracts was sufﬁcient to support a low level of DNA kinase activity (Figure 4D). Together, the experiments described above suggest that the mutant PNKP proteins present in MCSZ cells possess normal or near-normal levels of DNA phosphatase activity and also, in the case of PNKP E326K, DNA kinase activity, as measured in vitro at 30 C, but that these proteins are unstable in cells. While intracellular in- stability might be expected for the truncation mutants, it was more surprising for the point mutant PNKPE326K. To address this discrepancy, we examined the thermal stability of PNKP, PNKPL176F and PNKPE326K by pre-incubating the recombinant proteins at temperatures that spanned the physiological range (35–38 C) for a short period (2.5 min) prior to measuring their activity at 30 C. Consistent with the results above, PNKPE326K protein that was pre-incubated on ice exhibited near-normal DNA 30-phosphatase (Figure 5A, left panels) and DNA 50-kinase (Figure 5B, left panels) activity during