4772 Nucleic Acids Research, 2005, Vol. 33, No. 15 depend on a steric barrier for the 20-hydroxyl of an incoming rNTP (4,50,65). Accordingly, Polb and Poll are unable to incorporate rNTP since they have bulky residues close to the 20 position of the ribose of the incoming nucleotide (Tyr271-Phe272 and Tyr505-Phe506, respectively). However, Polm and TdT, which efﬁciently insert rNTPs (36,37,45), have a small residue in the pair (Gly433-Trp434 and Gly448-Trp449) that was shown to be responsible for rNTP insertion (34). As shown here, SpPol4 resembles Polm and TdT, as it also incor- porates rNTP very efﬁciently. This property was expected because the two residues equivalent to Polm (Gly433-Trp434) are strictly conserved in SpPol4 (Gly434-Trp435). Strikingly, it has been recently reported that ScPol4, although having two aromatic residue at these positions (His517-Tyr518) also incor- porates rNTPs with a high efﬁciency (64). It has been demonstrated that NHEJ is a predominant repair pathway in G1 phase and probably in non-cycling cells (66–68). In contrast to dNTPs, abundant during S phase, rNTPs are available at high levels in all phases of the cell cycle (69,70). Therefore, as suggested for human Polm (38) and ScPol4 (64), the extraordinary ability of SpPol4 to incorp- orate rNTPs would be very convenient for a role in NHEJ. Moreover, insertion of rNTPs might also be useful in BER to repair modiﬁed or damaged bases into DNA throughout the cell cycle that could be removed by the sequential action of RNaseH35/RNaseH type II and Rad27/FEN-1 (71). Further work should be carried out to ascertain this speciﬁc pathway in S.pombe. It has been reported that the imprinting step during mating- type switching in S.pombe is an RNase-sensitive modiﬁcation that consists of one or two RNA residues incorporated into the mat1 locus (55), which becomes a fragile chromosome site. Taking into account the capacity of SpPol4 to incorporate a few rNTPs in the DNA, it was tempting to speculate with a probable involvement of SpPol4 in mating-type switching in S.pombe. As shown here, the lack of SpPol4 (h90 pol4D strain) did not affect either the level of DSBs in the mat1 locus or the mating-type efﬁciency with respect to the h90 wild-type strain; therefore, we conclude that the imprint should remain unaf- fected. Thus, if there were some RNA residues in the mat1 locus, the incorporation would be SpPol4-independent. Alternatively, the imprint could imply a strand-speciﬁc nick with no ﬂanking RNA residues (72). In conclusion, the results presented here demonstrate that SpPol4 shares biochemical properties with different members of the PolX super-family; thus, it must be considered to be a unique enzyme (see Table 1 for a comparison). Mammalian PolXs became specialized to play a role in BER (Polb), in NHEJ coupled with BER (Poll), microhomology-mediated NHEJ (Polm) or V(D)J recombination (TdT). However, the fact that both ﬁssion and budding yeasts had only one DNA Pol X suggests that they are evolutionarily closer to the stem ancestor of the family, which is also consistent with a less specialized and multipotential role in different forms of DNA repair, enabled by a combination of the biochemical properties of their mammalian homologues. SUPPLEMENTARY MATERIAL Supplementary Material is available at NAR Online. ACKNOWLEDGEMENTS We thank Juan Jime´nez for S.pombe strains and plasmids and Aurelia Lahoz for her inestimable advice and help with S.pombe handling and techniques. This work was supported by Ministerio de Ciencia y Tecnolog´ıa Grant BMC 2003- 00186, and by an institutional grant to Centro de Biolog´ıa Molecular ‘Severo Ochoa’ from Fundacio´ n Ramo´ n Areces. 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