Nucleic Acids Research, 2012, Vol. 40, No. 14 6615 Figure 5. Thermal stability of WT and mutant recombinant histidine-tagged PNKP. (A) Thermal stability of PNKP DNA phosphatase activity. Top left, aliquots of the recombinant WT and mutant human PNKP (0.7 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 this panel were puriﬁed by IMAC alone. Middle and bottom, 30 and 60 nM of WT and the indicated mutant PNKP protein were pre-incubated for 2.5 min at the indicated temperatures and then subsequently incubated with 60 nM of the indicated oligonucleotide duplex substrate (top) for 30 min at 30 C. Reaction products were fractionated by denaturing PAGE and detected & by phosphorimaging as described in Figure 2. A representative gel is shown in the middle, and the quanti- ﬁcation from four independent experiments (±SEM) at the bottom. (B) Thermal stability of PNKP DNA kinase activity. Top and bottom, 30, 60 and 120 nM of WT and the indicated mutant PNKP protein was pre-incubated for 2.5 min at the indicated temperatures and then subsequently incubated with 60 nM of the indicated oligonucleotide duplex substrate (top) for 30 min at 30 C in the presence of [g-32P] ATP. Reaction products were fractionated by denaturing PAGE and detected and quantiﬁed by phosphorimaging as described in Figure 3. A representative gel is shown at the top and the quantiﬁcation (mean ± SEM) from four independent experiments at the bottom. different MCSZ mutations may impact on DNA strand break repair rates to a different extent, depending on the source and/or structure of the DNA break. DISCUSSION MCSZ is a human disorder associated with microcephaly, early-onset seizures and developmental delay (12). MCSZ results from mutations in PNKP, a DNA end-processing factor that possesses both DNA 30-phosphatase and DNA 50-kinase activities. To further understand the molecular defect behind MCSZ, the impact of the four disease- associated mutations identiﬁed to date on PNKP activity and rates of chromosomal DNA strand break repair was examined. When analysed at 30 C, the truncated MCSZ proteins PNKPT424Gfs48X and PNKPexon15 fs4X each exhibited levels of DNA 30-phosphatase activity comparable to