4764 Nucleic Acids Research, 2005, Vol. 33, No. 15 50-TGAAGTCCCTCTCGACTAAGAACCTGCACAGA-30; T13 (C), 50-AGAAGTGTATCTCGTACTCACTGTGA- TC-30; T18 (T), 50-ACTGGCCGTCGTTCTATTGTACT- CACTGTGATC-30; T4, 50-GTACCCGGGGATCCGTACG- GCGCATCAGCTGCAG-30; DG1, 50-AGATACACT- TCT-30; DG5, 50-AACGACGGCCAGT-30; D16, 50-GTCGA- GAGGGACTTCA-30; D15, 50-TCGAGAGGGACTTCA-30]. All the oligonucleotides were puriﬁed by 8 M urea–20% PAGE. Oligonucleotides SP1C, P15 (C), P15, P6 and oli- go(dT)15 were 50-labeled with [g-32P]ATP and T4 polynu- cleotide kinase. For dRP lyase activity assay, oligonucleotide P6 was 30-labeled with [a-32P]ddATP and TdT. Polymerase activity was evaluated by using synthetic double-stranded oligonucleotides as substrates. These sub- strates were prepared by annealing a 50-32P-end-labeled primer to different oligonucleotides to generate open (P15/T32 or SP1C/T18(T) and 1 or 2 nt gapped (P15/T32/D16; SP1C/ T13(C)/DG1 or P15/T32/D15) template/primer substrates in the presence of 0.2 M NaCl and 60 mM Tris–HCl, pH 7.5. The polymerization reactions were done in 12.5 ml of incubation mixture containing 50 mM Tris–HCl, pH 7.5, 2 mM MgCl2 or 1 mM MnCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, different concentrations of the indicated dNTPs or rNTPs, 4 nM of 50-labeled substrate and the indicated concentrations of puriﬁed hPolm, hPoll, SpPol4 or calf thymus TdT. After incubation for 15 min at 30 C, reactions were stopped by adding gel loading buffer [95% (v/v) formamide, 10 mM EDTA, pH 8, 0.1% (w/v) xylene cyanol and 0.1% (w/v) bromophenol blue]. Products were resolved and analyzed by denaturing 8 M urea–20% PAGE and autoradiography. Quantiﬁcation was done in a Fujix BAS1000. Cloning and purification of S.pombe DNA polymerase X Cloning of the S.pombe SpPol4 gene was started from the identiﬁcation of an open reading frame (ORF) (SPAC2F7.06c), in the public database S.pombe/GeneDB (http://www.genedb.org/) that codiﬁes for a putative DNA polymerase from the X family. Speciﬁc primers with restric- tion sites (in boldface) in their 50 ends SpPol4.50BgX (50-AGATCTTGCTCGAGCATGAAGATTCTTGCAAG- AAGATTCTTGCAAGCA-30) and SpPol4.30STOPNBg (50-AGATCTGCGGCCGCCCTATCCCGTGTTACGAAA- CTTT) were designed to amplify yeast genomic DNA. PCR was performed with Taq Expand High Fidelity (Roche) as follows: 35 cycles at 95 C for 30s, 50 C for 30s and 68 C for 120s. The 1551 bp SpPol4 PCR product was cloned in pGEM-T Easy (Promega) to generate plasmid pGEM-T Easy::SpPol4, veriﬁed by sequencing, digested with BglII and subcloned in the BamHI site of the expression vector pDS473a, which allows the expression of recombinant pro- teins as fusions with a glutathione S-transferase (GST)-tag, to generate the yeast expression plasmid pDS473-SpPol4. Expression of SpPol4 was carried out in the S.pombe wild- type strain sp7 transformed with plasmid pDS473-SpPol4. A 10 liters culture was grown at 30 C for 18–20 h in EMM supplemented with leucine (ﬁnal OD600 ¼ 1). Subsequently, the cultured cells were harvested at 4 C and washed with stop buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA and 1 mM NaN3, pH 8). The pelleted cells were weighted (10 g) and frozen ( 70 C). Just before puriﬁcation, which was carried out at 4 C, frozen cells were thawed on ice in the presence of 50 ml ice-cold lysis buffer [phosphate-buffered saline (PBS), 50 mM NaF, 2 mM EDTA, pH 8, 1% NP-40, 1.3 mM p-NH2-benzamidine, 1 mM phenylmethylsulfonyl ﬂuoride (PMSF) and 1 tablet of protease inhibitor cocktail; Roche] and broken with a French Press (twice at 20 000 psi). KCl was added to the lysate upto a ﬁnal concentration of 0.2 M. Cell debris were separated from the soluble lysates by ultra- centrifugation (50 000 g for 1 h at 4 C in a Beckman JA-25.50 rotor). The protein from the soluble fraction was subjected to afﬁnity chromatography. Gluthathione–Sepharose 4B (1 ml) (Pharmacia) packed into a column was equilibrated with 10 vol of lysis buffer. Soluble lysate was then loaded at 3 ml/h ﬂow rate into the column. Afterwards, the column was extensively washed with buffer IPP150 (PBS and 0.1% NP-40) and equi- librated with native binding buffer (100 mM Tris–HCl, pH 8 and 100 mM NaCl). After several washing steps with native binding buffer containing 20 mM gluthathione, polymerase- containing fractions estimated by Coomassie blue staining were collected, 2-fold diluted with buffer A (50 mM Tris–HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA and 1 mM DTT), and bound to a phosphocellulose column (1 ml) and eluted with buffer A containing 500 mM NaCl. This fraction contains highly puriﬁed GST-tagged SpPol4. Protein concen- tration was estimated by densitometry of Coomassie blue- stained 10% SDS–PAGE gels, using standards of known concentration. Under these conditions, the yield was 26 mg of puriﬁed GST-tagged SpPol4/g of S.pombe cells. This puriﬁed ﬁnal fraction, adjusted to 50% (v/v) glycerol and supplemented with 0.1 mg/ml BSA, was stored at 70 C. Construction and purification of a polymerization-deficient form of SpPol4 Site-directed mutations were introduced into pGEM-T Easy::SpPol4 plasmid by using a PCR-based method (QuickChange Site-Directed Mutagenesis kit; Stratagene) with the oligonucleotide 50-GCCTGTTGGAGCGGCCGTT- GCTATGGTGTTGAGTCC-30 and its reverse complementary oligonucleotide 50-GGACTCAACACCATAGCAACGGCC- GCTCCAACAGGC-30 for the double mutation D355A/ D357A. The plasmid pGEM-T Easy::SpPol4D355A/D357A gen- erated was sequenced and a BglII fragment containing the SpPol4D355A/D357A sequence was subcloned in the BamHI site of pDS473a. SpPol4D355A/D357A protein, which has two of the three catalytic aspartates mutated to alanines, was puriﬁed to homogeneity as the wild-type SpPol4 described above. DNA polymerization on activated DNA The incubation mixture contained, in 25 ml, 50 mM Tris–HCl, pH 7.5, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 13.2 nM [a-32P]dCTP, 1 mM (dATP, dCTP, dGTP, dTTP), 1 mM MnCl2 or 10 mM MgCl2 as metal activator, 625 ng of activated calf thymus DNA and 250 nM of the puriﬁed GST-tagged SpPol4 or SpPol4D355A/D357A. After incubation for 30 min at 37 C, the reactions were stopped by adding 10 mM TE/ 0.1% SDS and the samples were ﬁltered through Sephadex G-50 spin columns in 10 mM TE/0.1% SDS. The excluded volume, corresponding to the labeled DNA, was counted (Liquid Scintillation Counter; Pharmacia) and the