polymerization activity of SpPol4 was calculated as the amount of incorporated dCMP. 30!50 exonuclease assay The incubation mixture, in 12.5 ml, contained 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 50 nM SpPol4 and 1.5 nM single-stranded labeled P15 or P15/T32(C) hybrid. Reactions were incubated at 30 C for 15 min and were stopped by adding denaturing loading buffer. 30!50 exonucleolysis, expected to produce a degradation ladder of the labeled P15 primer, was analyzed by 8 M urea–20% PAGE and autoradiography. Electrophoretic mobility shift assay (EMSA) EMSA was performed using 1 [SP1C/T13(C)/DG1] and 5 nt [SP1C/T18(T)/DG5] gapped molecules to analyze the inter- action of SpPol4 and DNA. Gel mobility shift assays were performed in a ﬁnal volume of 12.5 ml containing 50 mM Tris–HCl, pH 7.5, 0.1 mg/ml BSA, 1 mM DTT, 4% glycerol, 4 nM labeled DNA and different concentrations of SpPol4 (250, 600 and 1200 nM). Samples were incubated for 10 min at 30 C to allow the formation of enzyme–DNA complexes. For competition analysis, 100 nM SpPol4 was incubated with labeled 1 nt gapped 50-phosphate molecules (4 nM) for 15 min at 30 C in a ﬁnal volume of 25 ml. After the complexes were formed, unlabeled 1 nt gapped 50-phosphate (200 and 600 nM) or 1 nt gapped 50-hydroxyl (200 and 600 nM) molecules were added to the reaction mixture and incubated for another 10 min at 30 C. After incubation, samples were mixed with 3 ml of 30% glycerol and resolved by native gel electrophoresis on a 4% polyacrylamide gel (80:1 monomer/ bis). After autoradiography, DNA polymerase–DNA com- plexes were detected as mobility retardation in the migration position of the labeled free DNA. Quantiﬁcation of the com- petition experiments was done in a Fujix BAS1000. The amount of the labeled GAP1-P forming SpPol4::GAP1-P complexes was calculated by normalizing the radioactive signal in the shifted band to the total radioactivity. dRP lyase activity assay As a substrate, the 30 end 34mer-labeled P6 oligonucleotide was annealed to the 34mer T4 oligonucleotide. This labeled double-stranded substrate (500 nM) was treated with UDG (100 nM) for 20 min at 37 C in buffer containing 50 mM HEPES, pH 7.5, 20 mM KCl and 2 mM DTT to remove the uracil. After incubation, the mixture was supplemented with 10 mM MgCl2 and 40 nM hAPE for 10 min at 37 C; thus, generating the substrate for dRP lyase activity. Reaction mixtures (25 ml) containing 50 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM KCl, 2 mM DTT, 70 nM concentration of the treated substrate and different amounts of either SpPol4 (20, 60 and 120 nM), SpPol4D355A/D357A (20, 60 and 120 nM), hPolm (70 nM) or hPoll (60 nM) were incubated at 37 C for 20 min. After incubation, NaBH4 was added to a ﬁnal concentration of 340 mM, and the reactions were kept for 20 min on ice. Stabilized (reduced) DNA products were ethanol precipitated in the presence of 0.1 g/ml of tRNA, resuspended in water and analyzed with 8 M urea–20% PAGE and visualized by autoradiography. Nucleic Acids Research, 2005, Vol. 33, No. 15 4765 In vitro reconstitution of BER A 34mer double-stranded DNA substrate that contained a G opposite uracil at position 16 was used. This molecule was treated as described above for the dRP lyase activity assay to generate the dRP-containing substrate. Reactions (25 ml) con- taining 70 nM substrate, 50 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM KCl, 2 mM DTT, 0.3 mM [a-32P]dCTP and either SpPol4 (125 nM) or human Poll (245 nM) were incu- bated for 20 min at 37 C. Later, each reaction was divided into two halves. One was incubated with 1 mM ATP and 40 U T4 DNA ligase for 10 min at 37 C and the other was mock- treated. Reactions were terminated by the addition of dena- turing loading buffer, analyzed by 8 M urea–20% PAGE and visualized by autoradiography. Genomic DNA preparation and imprint analysis in S.pombe Yeast chromosomal DNA was puriﬁed from a logarithmically growing culture (10 ml; OD600 0.5–1). Cells were harvested and resuspended in 200 ml breaking buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris–HCl, pH 8 and 1 mM EDTA), 200 ml glass beads and 200 ml phenol/chloroform/ isoamyl alcohol (25:24:1). After 15 min vortexing at high speed, 200 ml TE was added and the mixture was centrifuged for 5 min at 11 000 g. The aqueous layer was transferred to a clean tube and DNA was precipitated with ethanol. The pellet was resuspended in TE. HindIII-digested DNA (50 mg) was separated by agarose gel electrophoresis and analyzed by Southern hybridization using a 1 kb 32P-labeled mat1-P PCR probe. The oligonucleotides sequences used for the PCR were mat1-50, 50-AGAAGAGAGAGTAGTTGAAG- 30; and mat1P-30, 50-CCAATTCCTTCT-TGTATATGTTA- TAC-30. The mat2 (6.3 kb) and mat3 (4.2 kb) bands result from hybridization to the mat1 probe as they share cassette homology. The imprint could be converted into a DSB during standard methods of DNA puriﬁcation (43,44) and visualized by autoradiography. To determine the efﬁciency of mating- type switching, a standard iodine staining assay was carried out. Individual colonies were replicated onto EMM supple- mented with the appropriate amino acids and then were grown for 3 days at 22 C before being exposed to iodine vapors. RESULTS SpPol4 a unique X-family DNA polymerase in S.pombe The S.pombe ORF SPAC2F7.06c coding for a putative 736 amino acids DNA polymerase from the X family was over- produced in ﬁssion yeast wild-type cells and puriﬁed to near homogeneity as described in Materials and Methods. The pro- tein, expressed as a fusion protein containing a GST-tag at its N-terminus, was puriﬁed by gluthathione–Sepharose afﬁnity and phosphocellulose chromatography. After the puriﬁcation steps a unique polypeptide was observed in the ﬁnal fraction, identiﬁed by Coomassie blue staining after SDS–PAGE ana- lysis, migrating at the expected position for GST-tagged SPAC2F7.06c ( 84 kDa) (data not shown). The puriﬁed fraction was assayed for DNA polymerase activity on an acti- vated DNA. As expected, the puriﬁed fraction was able to catalyze dNTP incorporation in the presence of either