4768 Nucleic Acids Research, 2005, Vol. 33, No. 15 Figure 3. Characterization of SpPol4 template dislocation and primer realign- ment capacities at gapped DNA intermediates. Schemes representing template sequence contexts that are appropriate to evaluate slippage-mediated disloca- tion (A), dNTP selection-mediated dislocation (B), and primer realignment versus direct mismatch extension (C) are shown (see text for details). Labeled primer (asterisk) and 50 end phosphate group (P) are indicated. Transiently misaligned or mispaired nucleotides are indicated inside a circle, and nucleotide tracks inside a box. A correctly paired primer terminus is indicated inside a box. Polymerization assays were carried out as described in Materials and Methods using 250 nM either SpPol4, human Polm or human Poll, in the presence of each individual dNTP either at 10 mM in all cases (C), or at a different concentration for each DNA polymerase: Polm (500 nM), Poll (100 nM) and SpPol4 (5 mM) (A and B). Primer extension was analyzed by 8 M urea–20% PAGE and auto- radiography. Mobility of the unextended primer (P) and the 1 nt (+1) and the 2 nt (+2) extended primers are indicated at the autoradiographs. SpPol4 inserts both rNTPs and dNTPs with the same efficiency Polm and TdT have the striking ability to incorporate both rNTPs and dNTPs to nucleic acid chains (36,37,45). This unusual capacity mainly relies on a single glycine residue that opens the ‘steric gate’, which is frequently closed by a conserved aromatic residue present in Polb, Poll and in other members of the PolX family of DNA-dependent DNA poly- merases (Figure 4A) (36). Since SpPol4 also has a glycine residue at this position (Gly434), it seemed very likely that SpPol4 could incorporate rNTPs. By using the same four 1 nt gapped template–primer substrates as in Figure 2B, it was shown that SpPol4 efﬁciently incorporates rNTPs on a DNA primer strand (Figure 4B) with almost equal efﬁciency as dNTPs and displaying the same preference pattern for purines (compare Figures 4B and 2C). Using a competition assay in which both sugars (ribose and deoxyribose) are simultaneously provided, the sugar selectiv- ity factor of a given DNA polymerase can be calculated (36,45). Since the rNTP and the dNTP have different molecu- lar weights, the +1 extended primers can be easily separated by gel electrophoresis and quantiﬁed. The sugar selectivity factor is given as the ratio between the amounts of primer extended with rNTP versus dNTP. Irrespective of the nature of the base, the sugar selectivity factors obtained for SpPol4 were very similar (0.74–0.91) and proximal to one, indicating a lack of discrimination between rNTPs and dNTPs (Figure 4C). These values are similar to those obtained for TdT (43) and Polm (36) in untemplated and templated reactions, respec- tively, being several orders of magnitude smaller than the ones reported for other DNA polymerases (50–52). SpPol4 is not required for imprinting at the mat1 locus It is known for many years that mating-type switching in S.pombe depends on a strand-speciﬁc imprint at the mat1 locus (53,54). The imprint was characterized either as an alkali-labile modiﬁcation or as a nick that could be converted into a DSB during standard methods of DNA puriﬁcation (43,44). More recently, this imprint has been characterized as an RNase-sensitive modiﬁcation that consists of one or two RNA residues incorporated into the mat1 locus (55). Based on these results, it was tempting to speculate with the possibility that SpPol4 might be responsible at the incorp- oration of these one or two RNA residues. S.pombe genomic DNA was prepared by a standard yeast extraction protocol (see Materials and Methods), digested with HindIII and analyzed by Southern hybridization using a 1 kb mat1-P as a probe. The h90 wild-type strain yielded the typical bands of uncleaved (10.4 kb; mat1) and cleaved (5.4 kb; mat1*) products, together with two other bands representing cross- hybridization of the mat1-P probe with the mat2 (6.3 kb) and mat3 (4.2 kb) loci (Supplementary Figure 2A and B, lane 2). As expected, the smt-0 mutant strain (56), containing a dele- tion of the cis-acting elements SAS1 and SAS2 and thus preserving the integrity of the cleavage site sequence while abolishing mating-type switching, did not produce the mat1* band (57) (Supplementary Figure 2B, lane 1). However, the amount of cleavage at the mat1 locus, represented by the 5.4 kb (mat1*) band, obtained in the h90 pol4D mutant was the same as the level obtained in the h90 wild-type strain (Supplementary Figure 2B, compare lanes 2 and 3). Moreover, direct measure- ment of the mating-type switching efﬁciency by the iodine staining assay (see Materials and Methods) was carried out. The starch reaction with iodine vapors stains spore-containing colonies black, whereas slow-switching mutants exhibit streaky iodine staining patterns and colonies unable to switch