DNA in the presence of hUDG, hAPE and DNA ligase I (24,59,60). As shown in Figure 5C, two main products were observed in a human Poll-based reconstituted BER reac- tion: a 16mer product generated by a single nucleotide inser- tion (dCTP labeled) at the 30-hydroxyl end of the 50-incised AP site, and a 34mer product that corresponds to the complete repair of the DNA strand upon DNA ligase action. As shown in Figure 5C, both SpPol4 and hPoll are able to produce the same 16 and 34mer labeled products. Thus, SpPol4 is able to coord- inate both the gap-ﬁlling and dRP excision steps of repair preceding DNA ligase action. These data are consistent with a role for SpPol4 in BER and predict that the dRP lyase-containing enzyme SpPol4 could participate in BER in vivo. DISCUSSION In mammals, there are ﬁve members belonging to the X family of DNA polymerases: Polb, Poll, Polm, Pols and TdT. On the contrary, yeasts, plants, and some bacteria and viruses have only one PolX enzyme (3). In S.pombe, the entry SPAC2F7.06c (GeneDB http://www.genedb.org/) predicted a putative DNA PolX as inferred by sequence comparison analysis. Based on the results presented here, it can be con- cluded that SPAC2F7.06c does codify for a novel DNA poly- merase belonging to the PolX family that would be adequately designated as SpPol4. The structural organization of SpPol4 as an N-terminal BRCT domain followed by a C-terminal 39 kDa Polb-like core domain resembles members of the family X DNA poly- merases, such as Polm, TdT and Poll. Excluding the more variable N-terminal BRCT domain (61), SpPol4 is more clo- sely related to Polm (27% identical core residues), followed by Poll (24% identity) and Polb (20% identity). It is worth noting that budding yeast has also one DNA PolX (ScPol4) that, unlike SpPol4, resembles Poll in its core domain (25% iden- tity) and in its structural organization (62). Therefore, based only on their coding sequences, it was speculated that SpPol4 is a yeast orthologue of Polm, whereas ScPol4 would be an orthologue of Poll (3). As summarized in Table 1, our biochemical analysis demonstrated that SpPol4 is capable of carrying out DNA synthesis in a template-dependent manner and exhibits low processivity during primer extension. Such properties are shared by all members of the eukaryotic X family, except TdT [reviewed in (63)]. EMSAs showed that SpPol4 binds to 50-phosphate gapped substrates better than to those with Table 1. Comparison of SpPol4 properties to other template-dependent members of the DNA PolX family DNA PolX Stimulation Dislocation by Dislocation rNTP Terminal dRP by 50-P slippage by dNTP usage transferase lyase selection Yes Strong Strong Very strong Yes weak weak weak Strong weak Yes Yes No Yes No No ? weak Yes No Yes Yes Yes No Yes SpPol4 Yes ScPol4a No Poll Yes Polm Yes Polb Yes aTaken from (64). Nucleic Acids Research, 2005, Vol. 33, No. 15 4771 a 50-hydroxyl, and this should imply an improvement in polymerization on the former substrates. As indicated in Table 1, the improved polymerization activity dependent on a 50-phosphate group described here for SpPol4 is also an attribute of Polb and Poll (23), and Polm (R. Jua´rez, P. Andrade and L. Blanco, unpublished data), but not of ScPolIV (64). Most residues involved in dRP lyase activity are conserved between Polb and Poll (24). Among them, the nucleophile residue at position Lys72 (Polb) or Lys312 (Poll), responsible for 90% of the activity (18,24), is conserved in ScPol4 (Lys248), but not in Polm and TdT (which lack dRP lyase activity), and is also absent in SpPol4. Unexpectedly, SpPol4 was shown to have dRP lyase activity although it lacks this conserved residue. Nonetheless, other residues pro- posed in Polb to facilitate removal of the dRP group are indeed present in SpPol4, and an alternative lysine (Lys240) could be acting as the attacking nucleophile (for details see Figure 5). In any case, and based on our in vitro assays, we propose that SpPol4 could play a role in BER, as Polb and Poll. Based on the demonstration of an intrinsic dRP lyase activity, a similar role for ScPol4 has been proposed recently (64). As shown in this paper, the relative nucleotide usage of SpPol4 is different from that observed for other DNA- dependent DNA polymerases of the X family (Polb, Poll and Polm). In particular, SpPol4 preferentially inserts purine nucleotides in the following order: dG>>dA>dT>dC. Hydrolysis, alkylation, oxidation and deamination are the major forms of DNA damage in all living cells, which are mainly repaired by BER. It is worth noting that, at least in mammalian cells, purines are lost 20-fold more frequently than pyrimidines ( 10 000/cell/day versus 500/cell/day, respectively). Additionally, purines are the most frequently alkylated bases and guanine is the base more prone to oxidation, resulting in 8-oxoG (100–1000/cell/day) and along with adenine in a ring-opened form called formami- dopyrimidine (FaPyG and FaPyA). Only deamination, another prevalent form of DNA damage, occurs predominately at cyto- sine, turning it into uracil (100–500/cell/hour) (1,21). There- fore, it is tempting to speculate that the preference of SpPol4 for purine nucleotides has been adapted to cope with a more intensive role of repairing purine bases. In addition to its preference for small gaps, the unusual capacity of SpPol4 to accept misaligned template–primer molecules as a substrate and to realign 30-terminal mismatches would be very convenient for microhomology-mediated NHEJ. Moreover, some BER intermediates, as staggered nicks made by an AP endonuclease in opposite strands, origi- nate DSBs that would trigger the NHEJ pathway. Under these circumstances, a DNA repair polymerase endowed with dRP lyase activity would be very convenient to process the damaged DNA ends and eliminate the dRP residues. Physical and functional interactions with factors of NHEJ have been reported for Polm, Poll and ScPol4 (27,38,39,41), occurring through the BRCT domain of these proteins. The presence of a BRCT domain at the N-terminus of SpPol4 would support similar interactions with NHEJ factors operating in S.pombe. Most DNA polymerases have an exquisite sugar selectivity and prefer to incorporate dNTPs over rNTPs by a factor of 104- to 106-fold (50). Sugar discrimination has been shown to