and kinase domains form a structural unit that is resistant to trypsin digestion (15). In the WT enzyme, the structural unit is stabilized by anchoring of the kinase domain to the phosphatase domain via interactions between the C- terminal ﬁve residues (Q517–G521 in human PNKP) and the interdomain linker/phosphatase domain (15). Intriguingly, while F518 is lost in PNKPT424Gfs48X and PNKPexon15 fs4X, both mutants have acquired a new phenylalanine at their C-termini as a result of their frame-shift mutations (F469 in PNKPT424Gfs48X, which is 471 amino acids in length and F434 in PNKPexon15 fs4X, which is 436 amino acids in length). Perhaps these novel phenylalanine residues fulﬁl an analogous role to F518 in WT PNK, explaining their near-normal DNA phosphat- ase-speciﬁc activity at 30 C. Despite this, however, levels of PNKPT424Gfs48X and PNKPexon15 fs4X protein and phosphatase activity are greatly reduced in MCSZ cell extracts, most likely explaining the presence of disease pathology in affected individuals. Consequently, and since RT–PCR analysis failed to reveal nonsense mediated decay of PNKPT424Gfs48X or PNKPexon15 fs4X mRNA (12), we suggest that while PNKPT424Gfs48X and PNKPexon15 fs4X are stable at 30 C, they are unstable in cells and present at <10% of normal levels. In contrast to the truncated mutant proteins, the point mutant PNKPE326K exhibited near-normal or normal levels of both DNA 30-phosphatase and DNA 50-kinase activity, at 30 C. Despite this, the clinical presentation of MCSZ individuals harbouring the PNKPE326K mutation is no less severe than that of the other PNKP mutants (12). This discrepancy is likely explained by our ﬁnding that, similar to the truncation mutations, the E326K mutation results in 10 - to 20-fold reduced cellular levels of PNKP protein. Since reduced PNKPE326K levels in MCSZ do not appear to result from mRNA instability (12,27), it is likely that this point mutant results in signiﬁcant cellular PNKP instability. Consistent with this idea, while PNKPE326K exhibited near-normal DNA kinase and DNA phosphatase activity at 30 C, the activity of this mutant protein was signiﬁcantly reduced at 35–38 C. E326 forms hydrogen bonds with main chain amides of T323 and A334 and thereby helps correctly position the seventh a helix in the DNA phosphatase domain of PNKP (15,28). It is possible that lysine can be accommodated at this position at 30 C, but not at physiological temperature. Of the four MCSZ mutations identiﬁed to date, only PNKPL176F exhibited reduced levels of both DNA phos- phatase and DNA kinase activity, at 30 C. L176 is part of an important hydrophobic pocket within the DNA phosphatase domain and is located near to the catalytic aspartate, D171 (15,28). The benzyl side chain of phenyl- alanine would most likely not be accommodated within this pocket and thus cause localized disruption of the DNA phosphatase domain, which in turn may lead to mis-folding of the DNA kinase domain. Consistent with this idea, PNKPL176F was the only mutant PNKP protein that displayed altered chromatographic properties during puriﬁcation. This protein also exhibited signiﬁcant temperature sensitivity, with both its DNA phosphatase and DNA kinase activity greatly reduced by incubation for short periods at physiological temperature. It thus Nucleic Acids Research, 2012, Vol. 40, No. 14 6617 seems likely that, similar to the other MCSZ PNKP mu- tations, PNKPL176F levels will be reduced in MCSZ cells harbouring this mutation. However, evidence for this awaits the availability of an appropriate MCSZ cell line. It is currently unclear whether it is reduced DNA kinase or DNA phosphatase activity, or both, that accounts for MCSZ. All MCSZ mutations examined to date result in >10-fold reductions in PNKP protein level, suggesting that both DNA 50-kinase and DNA 30-phosphatase activities are greatly compromised in all MCSZ individ- uals. In addition, since we detected very little residual DNA kinase activity in recombinant PNKPT424Gfs48X or PNKPexon15 fs4X proteins, even at 30 C, we suggest that individuals harbouring these mutants are effectively for PNKP DNA 50-kinase activity. Consistent with this idea, we failed to detect any DNA 50-kinase activity in MCSZ cell extracts harbouring these truncations. In contrast, all four of the MCSZ mutations identiﬁed to date confer normal or near-normal levels of DNA phos- phatase activity in the recombinant proteins at 30 C and, where examined, detectable levels of DNA 30-phosphatase activity in protein extracts prepared from MCSZ cells. Consequently, while it is likely that PNKP DNA 30-phos- phatase activity is reduced >10-fold in all MCSZ cells, it is unlikely to be null. In agreement with their reduced cellular level of PNKP protein, all MCSZ cells examined to date exhibit reduced rates of chromosomal DNA strand break repair following g-irradiation, which induces DNA strand breaks of which 70% possess 30-phosphate termini. MCSZ individuals harbouring the PNKPT424Gfs48X and PNKPexon15 fs4X mutations also exhibited reduced rates of DNA strand break repair following treatment with camptothecin (CPT), a topoisomerase poison that induces SSBs har- bouring both 30-phosphate and 50-hydroxyl termini. Interestingly, MCSZ cells harbouring PNKPE326K exhibited normal DNA strand break repair rates following CPT. This may reﬂect the higher residual level of PNKP protein in MCSZ cell extracts harbouring this mutation, compared to other PNKP mutations (Figure 5A). It is worth noting that the reduced rate of DNA strand break repair observed in MCSZ cells following g-radiation most likely reﬂects reduced rates of SSB repair, because >95% of DNA breaks induced by g-rays are SSBs (2,29). However, it is important to note that PNKP is also implicated in the repair of DSBs (30–33). Similar to SSBs, the majority of DNA termini present at DSBs induced by ROS are 30-phosphate. MCSZ may thus result from defects in both SSBR and DSBR. Consistent with this idea, whereas microcephaly is a feature of the DSBR-defective disorders Lig4 syndrome and human immunodeﬁciency with microcephaly, seizures have been reported in patients with SCAN1 and in mice in which the SSBR protein XRCC1 is conditionally deleted in brain (11,34–37). In summary, we report that all of the MCSZ mutations examined to date result in 10-fold or greater reduced levels of PNKP protein, due most likely to reduced protein stability at physiological temperature. While two of the four mutations effectively ablate cellular PNKP DNA