Nucleic Acids Research, 2012, Vol. 40, No. 14 6613 Figure 3. DNA 50-kinase activity of recombinant PNKP harbouring mutations associated with MCSZ. (A and B) DNA 50-kinase activity of the indicated WT and mutant PNKP. 25 nM of the indicated substrate (top) was incubated without ( ) or with 10, 25, 50 or 100 nM of WT or the indicated mutant PNKP for 60 min at 30 C in the presence of [g-32P] ATP. Reaction products were fractionated by denaturing PAGE and detected by phosphorimaging. Note that in the substrate the 17-mer strand is labelled at the 50-terminus with 32P to provide an internal loading control. A representative gel is shown on the left and quantiﬁcation (mean ± SEM) from four independent experiments on the right. For quantiﬁcation, the signal intensity of the 17-mer and the 25-mer bands were quantiﬁed using Aida Image Analyser v.427 and the intensity of the 25-mer normalized by dividing by the intensity of the 17-mer. subsequent 30 min incubation at 30 C, whereas the activity of PNKPL176F was partially reduced. However, both PNKPE326K and PNKPL176F were more sensitive to pre-incubation at 35 C and 38 C than WT PNKP, with PNKPL176F exhibiting the greatest sensitivity (Figure 5A and B, middle and right panels). These data suggest that while the L176F and E326K mutations have relatively small effects on the speciﬁc activity of PNKPL176F and PNKPE326K at 30 C, they confer signiﬁcant instability to PNKP and reduced activity at physiological temperature. Finally, we compared the impact of MCSZ mutations on rates of chromosomal DNA strand break repair in living cells. To measure chromosomal DNA strand break repair rates we treated cells with g-radiation, which induces both SSBs and DSBs of which 70% harbour 30-phosphate termini, and measured total DNA strand breakage using alkaline comet assays. The MCSZ cell lines MC22601 and MC22602, harbouring PNKPT424Gfs48X and PNKPexon15 fs4X, exhibited reduced DNA strand break repair rates following g-radiation, as did the MCSZ cell line MC5403 harbouring PNKPE326K (Figure 6A and B). Since none of the MCSZ mutations disrupt the nuclear localisation of PNKP [(25) and Supplementary Figure 2], these experiments suggest that the reduced total levels of PNKP protein and activity in MCSZ cells are sufﬁcient to reduce the rate of chromo- somal DNA strand break repair. We also examined the impact of MCSZ mutations on the repair of DNA breaks induced by camptothecin (CPT), a topoisomerase I (Top1) poison that results in DNA strand breaks (26) possessing both 30-phosphate and 50-hydroxyl termini (17,20). Interestingly, while MC22601 and MC22602 MCSZ cells lines harbouring PNKPT424Gfs48X and PNKPexon15 fs4X accumulated elevated levels of DNA strand breaks during CPT treatment, MC5403 MCSZ cells harbouring PNKPE326K did not (Figure 5B and C). This suggests that