WT or mutant PNKP using GeneJuice (Novagen) accord- ing to the manufacturer’s instructions. After 24 h, trans- fected cells were ﬁxed with 4% paraformaldehyde and permeabilized with 0.5% triton. Nuclear DNA was counterstained with 40,60-diamidino-2-phenylindole (DAPI) and YFP positive cells were examined by ﬂuores- cent microscopy. Antibodies and immunoblotting Cells (2 105) were lysed in hot (90 C) SDS–PAGE loading buffer, fractionated by SDS–PAGE, and transferred to nitrocellulose for immunoblotting with anti-PNK polyclonal antibody (SK3195) at a concentration of 1/1000 in 1% milk overnight at 4 C. Alkaline comet assay LCLs (2 105 cells/sample) were mock treated (DMSO vehicle) or treated with 14 mM CPT for the indicated time periods at 37 C, or alternatively were mock irradiated or g-irradiated (20 Gy) on ice and incubated at 37 C for the indicated repair periods. Chromosomal DNA strand breaks were then quantiﬁed using alkaline comet assays and Comet Assay IV software (Perceptive Instruments) as previously described (21). RESULTS To date, four PNKP mutations have been identiﬁed in MCSZ (Figure 1) (12). To analyse the impact of these mu- tations on the DNA 30-phosphatase and DNA 50-kinase activities of PNKP, recombinant His-PNKP containing the MCSZ mutations was expressed in E. coli and puriﬁed by cation exchange chromatography and/or immobilized metal chelate afﬁnity chromatography (Figure 2A and B; Supplementary Figure S1). We noted that whereas PNKPE326K, PNKPT424Gfs48X and PNKPexon15 fs4X exhibited similar chromatographic properties to WT PNKP during puriﬁcation, PNKPL176F failed to bind a cation exchange column (Supplementary Figure S1). As a negative control for DNA phosphatase activity, we also expressed and puriﬁed in parallel the protein PNKPD171N, which harbours a mutation in the DNA phosphatase catalytic domain and retains little or no DNA phosphatase activity (15,22,23). To measure DNA 30-phosphatase activity we employed a 32P-radiolabelled oligonucleotide duplex that harboured a 1-bp gap with 30-phosphate and 50-phosphate termini (Figure 2C, top). Strikingly, all of the MCSZ mutant PNKP proteins exhibited signiﬁcant 30-phosphatase activity on this substrate, which mimics a common type of oxidative SSB, as measured by their ability to reduce the electrophoretic mobility of the 32P-labelled 17-mer (Figure 2C and D). Indeed PNKPE326K, PNKPT424Gfs48X and PNKPexon15 fs4X each possessed levels of DNA 30-phos- phatase activity comparable to WT PNKP (Figure 2C, right), whereas PNKPL176F exhibited a modest ( 50%) reduction in this activity (Figure 2D). In contrast, and in agreement with previous studies, PNKPD171N exhibited greatly reduced DNA 30-phosphatase activity (Figure 2C and D) (22,23). To measure DNA 50-kinase activity we Nucleic Acids Research, 2012, Vol. 40, No. 14 6611 employed a 32P-radiolabelled oligonucleotide duplex con- taining 30-hydroxyl and 50-hydroxyl termini (Figure 3A, top). Phosphorylation of the 50-terminus of this substrate by recombinant PNKP, in the presence of [g-32P] ATP, was indicated by the appearance of a 32P-labelled 25-mer product (Figure 3A and B, left). Note that the pre-labelled 32P-17-mer in this substrate served as an internal loading control. As expected, and in agreement with previous studies, PNKPD171N possessed normal levels of DNA 50-kinase activity (Figure 3A and B) (22–24). In contrast, PNKPT424Gfs48X and PNKPexon15 fs4X, which contained large deletions within the DNA kinase domain, possessed very little DNA 50-kinase activity (Figure 3A). DNA 50-kinase activity was also signiﬁcantly reduced in PNKPL176F, despite this mutation being located in the DNA phosphatase domain (Figure 3B). However, PNKPE326K displayed near-normal levels of DNA 50- kinase activity (Figure 3A). Next, we examined the impact of MCSZ mutations on PNKP activity in whole-cell extracts prepared from MCSZ lymphoblastoid cells (LCLs). For this study, LCLs from three MCSZ patients and three unaffected relatives were available for analysis (Figure 1B) [previously described in (12)]. MC22601 and MC22602 are LCLs from MCSZ siblings that are compound heterozygotes for the mutations T424Gfs48X and exon15 fs4X, and MC22604 and MC22606 are LCLs from unaffected relatives that are heterozygous for the mutation exon15 fs4X. MC5403 is from an individual with MCSZ that is homozygous for the E326K mutation, and MC5405 is from a WT unaffected relative. Cells from MCSZ individuals harbouring PNKPL176F are currently not available. In agreement with our previous report, all of the LCLs from individuals with MCSZ possessed greatly reduced levels of PNKP protein, retaining 5–10% of the normal level (Figure 4A) (12). Consistent with this, the three MCSZ cell extracts exhibited greatly reduced 30-phosphatase activity on single-stranded and duplex DNA substrates at 30 C, as measured by the appearance of dephosphorylated (17-OH) product, compared to extracts from WT or heterozygous individuals (Figure 4B and C). Notably, however, all of the MCSZ extracts possessed some phosphatase activity, as suggested by the appearance of an 18-mer product in reactions con- taining duplex substrate and high concentrations of mutant cell extract, which reﬂected DNA polymerase activity on the nascent 30-hydroxyl termini and ﬁlling of the 1-bp gaps (Figure 4C). To measure DNA 50-kinase activity in LCL extracts we employed an oligonucleotide duplex harbouring a nick with a 50-hydroxyl terminus present on a 25-mer (Figure 4D, top). Phosphorylation of the 50-hydroxyl terminus on the 25-mer was measured indirectly in these experiments, by the appearance of a 43-bp product resulting from ligation of the 32P-labelled 17-mer and the phosphorylated 25-mer. We failed to detect any DNA ligation product in reactions containing MCSZ cell extract from either MC22601 or MC22602 LCLs (Figure 4D), consistent with our observa- tion that the mutant PNKPT424Gfs48X and PNKPexon15 fs4X proteins present in these cell extracts retain very little DNA kinase activity. In contrast, a small amount DNA ligation product was observed in reactions containing MCSZ cell