involved in the repair of damaged nucleotides (19,20). This multistep process is initiated with the removal of the modiﬁed base by a speciﬁc DNA N-glycosylase yielding apurinic/ apyrimidinic (AP) sites. AP sites are recognized and incised by an AP endonuclease leaving single strand breaks (SSBs) with a 50-dRP end. Finally, the 50-dRP can be released either by the 50-dRPase activity of Polb (short-patch BER) or by the combined action of a DNA polymerase (Polb, and Pole or Pold) and the 50-ﬂap endonuclease FEN1 (long-patch BER) (21,22). Poll has 32% amino acid identity to Polb and contains an intrinsic dRP lyase activity that can substitute for Polb in BER in vivo and in vitro (23,24). However, the high afﬁnity of Poll for deoxynucleotides (dNTPs) (37-fold over Polb) is consis- tent with its possible involvement in DNA transactions occur- ring under low cellular levels of dNTPs, i.e. in non-replicating phases of the cell cycle (25). Similar to Polb, Poll inserts dNTPs in a DNA template-dependent manner and is proces- sive in small gaps containing a 50-phosphate group (25). In addition, immunodepletion of nuclear extracts of HeLa cells (26) and recent studies in which Poll associates with a Ku- XRCC4–DNA ligase IV–DNA complex (27–29) suggest a possible role for Poll in non-homologous end-joining (NHEJ). Polm has 41% identity to TdT, a template-independent DNA Pol X responsible for the N-addition during V(D)J recomb- ination of the immunoglobulin genes and T-cell receptor genes (30,31). Polm-deﬁcient mice are impaired in V(D)J recomb- ination of the immunoglobulin k light chain (32), which is initiated by an induced double strand break (DSB) that is repaired by an NHEJ mechanism, similar to that employed by other tissues to repair DSBs. However, unlike TdT, whose expression pattern is restricted to lymphoid tissues, Polm is expressed in additional tissues (33), suggesting a more general role of Polm in DNA repair (34). Polm behaves as an error-prone DNA polymerase, since it is able to induce/accept dislocations of the template strand (35). Unlike Polb and Poll, Polm is able to insert ribonucleotides (rNTPs) to a DNA chain (36,37) and lacks dRP lyase activity (24). Based on these properties and on the physical and func- tional interactions with the Ku-XRCC4–DNA ligase IV–DNA complex (38), it has been proposed that Polm functions in NHEJ and V(D)J recombination by promoting microhomol- ogy search and pairing activities (27,29). Polm is not a strictly template-dependent DNA polymerase, since it has an intrinsic terminal transferase activity (33) that probably plays a role in microhomology-mediated NHEJ reactions (R. Jua´rez, J. F. Ruiz, S. A. Nick McElhinny, D. A. Ramsden and L. Blanco, manuscript submitted). In contrast to mammals, budding and ﬁssion yeasts have only one DNA PolX enzyme (3). Whereas ScPol4 is closely related to Poll (25% amino acid identity to the Poll core), the putative DNA PolX from the ﬁssion yeast Schizosaccharomyces pombe (SPAC2F7.06c) is more closely related to Polm than to Poll (27% versus 24% identical core residues, respectively). ScPol4 was the ﬁrst DNA PolX shown to play a role in NHEJ (39). In agreement with that, it has been shown to have a direct interaction of the BRCT domain of ScPol4 with the Lig4/Lif1 complex (40), and a physical and functional interaction of Rad27 with both ScPol4 and Dnl4/Lif1 (41). No functional data have been reported for the putative DNA PolX (SPAC2F7.06c) from the ﬁssion Nucleic Acids Research, 2005, Vol. 33, No. 15 4763 yeast S.pombe, a unicellular eukaryotic organism whose properties closely resemble those of higher eukaryotic organ- isms. For this reason, S.pombe is a good model system for the analysis of gene products involved in DNA repair. Here, we report the cloning, expression and biochemical characteriza- tion of the SPAC2F7.06c gene product from S.pombe. DNA polymerization properties and the presence of a dRP lyase activity support a role of this DNA polymerase in both NHEJ and BER reactions. In spite of the closer similarity to Polm, this enzyme combines Polb, Polm and Poll properties, and therefore, it should be more unambiguously referred to as SpPol4. MATERIALS AND METHODS Strains and growth conditions Cells were grown at 30 C in rich medium (YES; 0.5% yeast extract, 3% glucose and supplemented with 200 mg/l of leucine and uracil) or in minimal medium (EMM). Appropri- ate amino acids and thiamine were added to EMM when required to a ﬁnal concentration of 200 mg/l and 25 mM, respectively. Geneticin selection was performed using YES medium containing 100 mg/l G418 (Sigma). The pol4D::KanMX strains, sp8 and sp10, were created from the wild-type strains, sp7 (h- leu1-32 ura4D18) and sp968 (h90), respectively, using the PCR-based method and the primers, pol4.A (50-TCCCTTAGTTGTAATTGTTCAAAATGAAG- ATTCTTGCAAGCAGCTGAAGCTTCGTACGCT-30) and pol4.B (50-AGTAATGTGGCGATCTTAAGGTCAAGATA- G-GTATTTACTACTAGTGGATCTGATATCATC-30). The nucleotide sequences in boldface overlap to the KanMX cassette of plasmid pFA6a-kanMX4 (42). The deletion was conﬁrmed by PCR using primers pol4.C (50-AGATCTGTT- CAAAATGAAGATT-CTTGC-30) and pol4.D (50-CTGCA- GAGTAATGTGGCGATCTTA-AGG-30) and by Southern blot (data not shown). Nucleotides and proteins Ultrapure unlabeled dNTPs and rNTPs, [g-32P]ATP, [a-32P]dCTP and [a-32P]ddATP (3000 Ci/mmol) were pur- chased from Amersham Biosciences. T4 polynucleotide kinase, UDG and T4 DNA ligase were purchased from New England Biolabs; TdT was obtained from Promega; restriction endonucleases and Taq Expand High Fidelity were obtained from Roche; hAPE was a gift from Dr S. H. Wilson (NIEHS, Research Triangle Park, NC). Puriﬁed human Poll and Polm were obtained as described previously (25,33). Oligonucleotides, templates and substrates for DNA polymerization Synthetic DNA oligonucleotides were obtained from Invitrogen [P15, 50-TCTGTGCAGGTTCTT-30; P15 (C), 50-TCTGTGCAGGTTCTC-30; SP1C, 50-GATCACAGTGA- GTAC-30; P6, 50-CTGCAGCTGATGCGCUGTACGGATC- CCCGG-GTAC-30; T32 (A), 50-TGAAGTCCCTCTCGAC- AAAGAACCTGCACAGA-30; T32 (C), 50-TGAAGTCCCT- CTCGACCAAGAACCTGCACAGA-30; T32 (G), 50-TGAA- G-TCCCTCTCGACGAAGAACCTGCACAGA-30; T32 (T),