6610 Nucleic Acids Research, 2012, Vol. 40, No. 14 Puriﬁcation of recombinant proteins His-tagged recombinant PNKP was expressed in the Escherichia coli strain BL21 (DE3) for 90 min at 20 C following the addition of 1 mM IPTG. Cells were sonicated in 50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 10% glycerol, 1 mM DTT, 1 mM imidazole (pH 8.0), 1 mM PMSF and clariﬁed by centrifugation. His-PNKP was puriﬁed by immobilized metal chelate chromatog- raphy (IMAC) using Ni–NTA Agarose resin (Qiagen), washing successively with wash buffer [50 mM Tris–HCl (pH 7.5), 0.5 M NaCl, 10% glycerol, 1 mM DTT] contain- ing either 20 mM (10 CV), 40 mM (10 CV) or 80 mM (10 CV) imidazole, and eluting in wash buffer containing 250 mM imidazole. Elution fractions containing His-PNKP were pooled, EDTA added to 10 mM to chelate eluted nickel ions (19), and dialysed in 50 mM Tris–HCl pH 7.5, 0.1 M NaCl, 10% glycerol, 1 mM DTT. With the exception of His-PNKPL176F, His-PNKP was then further puriﬁed by cation exchange chromatog- raphy and a 0.1–1 M NaCl gradient on a Mono S 5/50 GL column and A¨KTA Puriﬁer UPC 10 system (GE Healthcare). Finally, elution fractions containing recom- binant PNKP were dialysed [25 mM Tris–HCl, 150 mM NaCl, 10% glycerol, 1 mM DTT], aliquoted and snap frozen in liquid nitrogen and stored at 80 C. Preparation of oligonucleotide duplex substrates Oligonucleotide duplexes containing a SSB with a 1-bp gap and either 30- and 50-phosphate termini or 30- and 50-hydroxyl termini were prepared by annealing a 17-mer oligonucleotide (50-TCCGTTGAAGCCTGCTT-30) harbouring a 30-phos- phate or 30-hydroxyl terminus (as appropriate) and a 25-mer oligonucleotide (50-GACATACTAACTTGAGCG AAACGGT-30) harbouring a 50-phosphate or 50-hydroxyl terminus (as appropriate) with a 43-mer (50-CCGTTTCGC TCAAGTTAGTATGTCAAAGCAGGCTTCAACGG AT-30). Oligonucleotides were purchased from MWG- Biotech AG and the 17-mer was phosphorylated with phosphatase-dead T4 PNK (Roche) and [g-32P] ATP. Preparation of human lymphoblastoid cell-free extracts Total cell extracts were prepared from 7.5 106 cells by lysis in 300 ml 20 mM Tris–HCl (pH7.5), 10 mM EDTA, 1 mM EGTA, 100 mM NaCl, 1% triton-X100 and 1/100 dilution of protease inhibitor cocktail (Sigma). Extracts were clariﬁed by centrifugation at 14 000 rpm for 20 min at 4 C, aliquoted and snap frozen. Protein was quantiﬁed with a Bio-Rad DC Protein Assay Kit using BSA as a standard. In vitro DNA phosphatase and DNA kinase reactions For DNA phosphatase reactions employing recombinant PNKP, 25 nM of the indicated 32P-labelled substrate was incubated with the indicated concentrations of recombin- ant protein for 1 h at 30 C in 25 mM Tris–HCl pH 7.5, 130 mM KCl, 10 mM MgCl2 and 1 mM DTT. Reactions were stopped by addition of one-third volume of formamide gel-loading buffer and reaction products fractionated by 15% denaturing PAGE and analysed by phosphorimaging. PNKP phosphatase activity was quantiﬁed by measuring the signal intensity of the 17-P (substrate) and 17-OH (product) bands using Aida Image Analyser v.427 and calculating the percentage of substrate converted to product. For DNA kinase reactions employ- ing recombinant PNKP, 25 nM of the indicated 32P-labelled substrate was incubated with the indicated concentrations of recombinant protein for 1 h at 30 C in 25 mM Tris–HCl pH 7.5, 130 mM KCl, 10 mM MgCl2, 50 nM ATP, 25 nM [g-32P] ATP (6000 Ci/mmol) and 1 mM DTT. Reactions were stopped and products analysed as described above. PNKP kinase activity was quantiﬁed by phosphorimaging by normalizing the signal intensity of the labelled 25-mer (product) with the signal intensity of 32P-labelled 17-mer internal control. For reactions employing cell-free extract, reactions con- tained the indicated amounts of extract and 25 nM of the indicated 32P-labelled substrates and were incubated for the indicated time at 30 C in 25 mM Tris–HCl pH 7.5, 130 mM KCl, 1 mM DTT, 10 mM MgCl2. For reactions in which DNA gap ﬁlling or DNA ligation was measured, 100 mM dNTPs or 1 mM ATP, respectively, was included in the reaction buffer. A total of 1000-fold molar excess of a competitor oligonucleotide of unrelated sequence (50-TC TGCTAGCATCGATCCATG-30) and harbouring a 50-phosphate and 30-hydroxyl terminus was also included to reduce degradation of 32P-labelled substrate by non- speciﬁc nucleases. Reactions were stopped and products analysed as described above. For thermal inactivation DNA phosphatase assays, the indicated concentrations of recombinant PNKP were pre-incubated for 2 min 30 s at the indicated temperatures and then incubated for 30 min with 60 nM of the appropriate 32P-labelled DNA phosphatase substrate as described above. For thermal inactivation DNA kinase assays, the indicated concentrations of PNKP were pre-incubated as above at the indicated temperatures and then incubated with 60 nM of the appropriate 32P-labelled DNA kinase substrate for 30 min at 30 C in 25 mM Tris–HCl pH 7.5, 130 mM KCl, 10 mM MgCl2, 50 nM ATP, 25 nM [g-32P] ATP (6000 Ci/mmol) and 1 mM DTT. Reactions were stopped and products analysed and quantiﬁed as described above. Cell lines and cell culture MC22601 and MC22602 are MCSZ lymphoblastoid cells (LCLs) and are compound heterozygous for the mutations T424Gfs48X and exon15 fs4X. MC22604 and MC22606 are LCLs from unaffected relatives and are heterozygous carriers of the mutation exon15 fs4X. MC5403 is a MCSZ LCL and is homozygous for the E326K mutation and MC5405 is a WT LCL from an unaffected relative. The 1635 is a LCL from an individual with SCAN1 and AG09387 is a WT LCL from an unrelated individual. MC22601, MC22606, MC22604, MC22606, MC5403 and MC5405 (12) and 1635 (20) have all been described previously, and AG09387 was kindly provided by Dr Mark O’Driscoll. Flourescence microscopy A549 cells were seeded onto coverslips (1 104/cm2) and transfected 48 h later with pEYFP constructs encoding