processing. There are notable differences between these disorders, however. For example, whereas MCSZ is pri- marily a developmental disease, associated with primary microcephaly and seizures from an early age, AOA1 and SCAN1 are progressive, degenerative, disorders. MCSZ is the most recent disease identiﬁed in which the rate of SSBR is reduced. PNKP, the protein mutated in MCSZ, is a bi-functional enzyme that possesses both DNA 30-phosphatase and DNA 50-kinase activities. PNKP is a modular protein that possesses three distinct domains; an amino-terminal fork-head-associated domain (FHA) that mediates interactions with the SSBR and double-strand break repair (DSBR) scaffold proteins XRCC1 and XRCC4, respectively and the DNA phosphat- ase and DNA kinase catalytic domains (13–15). PNKP is a component of both SSBR and DSBR pathways and most likely operates at the majority of DNA breaks arising from attack of deoxyribose by ROS, because 50–70% of such breaks harbour 30-phosphate termini (2,6). In addition, DNA strand breaks resulting from abortive topoisomer- ase 1 (Top1) activity or created by bi-functional DNA glycosylases during DNA base excision repair can also harbour 30-phosphate termini (6,16,17). To date, four mutations in PNKP have been identiﬁed in MCSZ (12). L176F and E326K are point mutations of highly conserved residues within the DNA phosphatase domain and T424Gfs48X and exon15 fs4X are frame-shift mutations in the DNA kinase domain that result in premature stop codons and truncated PNKP polypeptides (Figure 1). T424Gfs48X is the result of a 17-bp duplication within exon14 in the kinase domain, resulting in a 471 amino acid polypeptide of which the last 48 residues are out-of-frame. Exon15 fs4X results in a 17-bp deletion within intron 15 that leads to mRNA transcript lacking exon 15, resulting in a polypeptide of 436 amino acids of which the last four residues are out-of-frame (12). E326K and T424Gfs48X are the commonest MCSZ mutations reported to date, with seven individuals homozygous for E326K and three homo- zygous for T424Gfs48X. Additionally, four MCSZ individ- uals possessing the T424Gfs48X mutation are compound heterozygotes, harbouring the additional mutations L176F or exon15 fs4X. Here we have analysed the impact of the MCSZ mutations on the speciﬁc activities of PNKP, to establish the molecular basis of this disease. MATERIALS AND METHODS DNA constructs pET16b–PNKP encoding His-tagged human PNKP has been described previously (13). pET16b–PNKPD171N encodes a His-tagged phosphatase deﬁcient PNKP mutant that was sub-cloned from pCD2E-PNKD171N (18). pET16b– PNKPL176F, pET16b–PNKPE326K, pET16b– PNKPT424Gfs48X and pET16b–PNKPexon15 fs4X encode His-tagged PNKP proteins harbouring MCSZ mutations. pET16b–PNKPL176F and pET16b–PNKPE326K were created by site-directed mutagenesis of pET16b–PNKP using a QuikChange XL mutagenesis kit (Agilent Technologies) and the primer sets [50-ATCTGGACGGGA Nucleic Acids Research, 2012, Vol. 40, No. 14 6609 Figure 1. PNKP mutations in MCSZ. (A) Schematic depicting the four PNKP mutations identiﬁed in MCSZ, to date. Note that two point mutations (L176F and E326K) are located in the DNA phosphatase domain and two frame-shift mutations (T424Gfs48X and exon15 fs4X), resulting in premature stop codons and truncated PNKP protein, are located in the DNA kinase domain. FHA denotes the fork-head-associated domain. (B) PNKP genotype of known MCSZ families, unaffected relatives, and associated LCLs used in this study. All individuals and families have been described previously (12). CGTTCATCACCACACGC-30/50-GCGTGTGGTGAT GAACGTCCCGTCCAGAT-30; pET16b–PNKPL176F] and [50-CTTCGCCACGCCTGAGAAGTTCTTTCTCA AGTG-30/50-CACTTGAGAAAGAACTTCTCAGGCG TGGCGAAG-30; pET16b–PNKPL176F], respectively. pET16b–PNKPT424Gfs48X and pET16b–PNKPexon15 fs4X were generated by RT–PCR ampliﬁcation of the PNKP region spanning the 17-bp duplication in T424Gfs48X (using the primers 50-AAA GAC TTC TCC TGC GCC GAT CG-30 and 50-CTC CAC CCA TAG CCG GAA CG-30) and the region deleted from exon15 in exon15 fs4X (using the primers 50- AAA GAC TTC TCC TGC GCC GAT CG-30 and 50-TTT GGA TCC TTC AGC CCT CGG AGA ACT GGC A-30), using mRNA extracted from the compound heterozygote MCSZ lymphoblastoid cell line, MC22602. The PCR frag- ments harbouring the truncation mutations were then sub-cloned into pET16b–PNKP using SacI/AgeI (pET16b–PNKPT424Gfs48X) and SacI/BamHI (pET16b– PNKPexon15 fs4X). pEYFP-PNKP, pEYFP-PNKPL176F, pEYFP-PNKPE326K, pEYFP-PNKPT424Gfs48X and pEYFP-PNKPexon15 fs4X are mammalian expression vectors encoding YFP-tagged wild-type (WT) and mutant PNKP and were created by PCR ampliﬁcation of the PNKP ORF in pET16b–PNKP, pET16b–PNKPL176F, pET16b–PNKPE326K, pET16b–PNKPT424Gfs48X and pET16b–PNKPexon15 fs4X, respectively, using the primers 50-AAA GAT CTA AAA TGG GCG AGG TGG AGG CC-30 and 50-AAA AGC TTA AAT CAG CCC TCG GAG AAC TGG C-30 and sub-cloning the ampliﬁed products into pEYFP-C1 (Clontech) using BglII/HindIII.