and catalysis. The formation of stable SpPol4/DNA com- plexes, assessed by EMSA, required a lower enzyme concen- tration when the 1 nt gapped DNA had a 50-phosphate group (Figure 1C). Even more, the afﬁnity of SpPol4 for the 50-phosphate group is so strong that when the primer strand is removed, SpPol4 still binds this molecule almost with the same efﬁciency (data not shown). To further analyze the stabilizing effect of the 50-phosphate on DNA binding by SpPol4, competition analysis were carried out as indicated in Materials and Methods. As expected, the amount of SpPol4::GAP1-P (labeled) complexes formed in a previous step progressively decreased when increasing amounts of unlabeled competitor DNA (either having a 50-phosphate or not) were added, being greater the competition with unlabeled GAP1-P (Figure 1D). However, even at 150- fold molar excess of the 1 nt gapped 50-phosphate competitor, the amount of SpPol4::GAP1-P labeled complexes was reduced only by 5% in comparison with that in the absence of the competitor (Figure 1D). Taken together, these results clearly demonstrate that SpPol4 binds stably and preferentially to a 50-phosphate containing DNA gap. SpPol4 is a template-instructed polymerase with preference for purines We evaluated the ability of SpPol4 to discriminate among the four dNTPs in order to catalyze template-directed DNA syn- thesis. We used a set of 1 nt gapped template-primer substrates with each of the four (X ¼ A, C, G or T) bases as template and having a 50-phosphate ﬂanking the gap (Figure 2A). For each substrate, the four dNTPs, one complementary to the template and the other three non-complementary, were assayed indi- vidually at different concentrations. As shown in Figure 2B, on the four 1 nt gapped substrates, SpPol4 preferentially incor- porated the nucleotide complementary to the ﬁrst template base, even when non-complementary nucleotides were pro- vided at a 100-fold higher concentration. Therefore, these results suggest that SpPol4 is template-instructed, i.e. it per- forms DNA synthesis following the Watson–Crick base pair- ing rules. Interestingly, quantiﬁcation of the efﬁciency of incorporation of each complementary dNTP demonstrated a strong imbalance in correct dNTP incorporation with prefer- ence for purines: dG>>dA>dT>dC (Figure 2C). Template dislocation and primer realignment capacities of SpPol4 Human Polm behaves as an error-prone DNA polymerase, since it is able to induce/accept dislocations of the template strand (35), which is likely crucial for its NHEJ function. To examine whether SpPol4 is similarly error-prone, we analyzed DNA synthesis in some template sequence contexts that are appropriate for evaluating: (i) slippage-mediated dislocation (Figure 3A); (ii) dNTP selection-mediated dislocation (Figure 3B); and (iii) primer realignment versus direct mis- match extension (Figure 3C). Each dNTP was provided indi- vidually to identify the opted mechanism for each DNA polymerase. In the substrate with the dA-track repeat, normal DNA synthesis would lead to dT incorporation, whereas slip- page of the primer terminus (dT) to the next template base (dA) would result in dG incorporation and a 1 frameshift DNA synthesis (see schematic representation in Figure 3A). Nucleic Acids Research, 2005, Vol. 33, No. 15 4767 Figure 2. SpPol4 preferentially incorporates complementary nucleotides. (A) Scheme representing the set of 1 nt gapped substrates with a 50 phosphate group (P) used in this assay, only differing in the templating base (X). The primer strand was 50 end labeled (asterisk). The oligonucleotides used to obtain these substrates were T32 (A, C, G or T)/P15/DG16. (B) Single nucleotide gap-filling assays using any of the four 1 nt gapped DNA substrates, and the four dNTPs (dA, dC, dG and dT) individually provided. Reactions were carried out as described in Materials and Methods using 125 nM SpPol4. Extension of the labeled primer strand in the presence of either the correct (1 mM) or the incorrect (100 mM) dNTP was analyzed by 8 M urea–20% PAGE and autoradiography. (C) Quantification of the complementary dNMP incorporation for the four 1 nt gapped molecules at different dNTP concentrations. The values plotted repre- sent the ratio between the amounts of extended versus total primers, and are the mean of four independent experiments. Unlike Polm, which clearly preferred to insert dG by a slippage-mediated dislocation mechanism, SpPol4 and Poll predominantly incorporated dT (Figure 3A). However, both polymerases also incorporated dG and therefore, they can misalign the template-primer to some extent. As shown in Figure 3B, changing the third dA of the track for a dG reduces the possibility of template slippage; therefore, DNA synthesis is more restricted to the canonical incorporation of dC, like SpPol4 and Poll do. Besides this normal DNA incorporation event, only Polm was also able to insert the complementary base (dG) to the position +2 in the template (dC). As reported earlier for Polm, the template dislocation requirement would be stabilized by the incoming dNTP (49). To examine the mismatch extension capacity of SpPol4, we performed a primer extension assay starting from a dA:dC base pair mismatch (see schematic representation in Figure 3C). Because SpPol4 and other PolX enzymes cannot remove mismatched nucleotides at the primer 30 end, only two outcomes are possible: (i) direct mismatch extension inserting dC; and (ii) primer terminus realignment inserting dG. As can be seen in Figure 3C, Poll uses both alternatives almost equally well. In agreement with its extreme error-proneness, Polm is able to extend the mismatch with any of the four dNTPs. Interestingly, SpPol4 has a more restricted behavior, as it is only able to insert dG, indicating a signiﬁcant primer realignment capacity that enables this enzyme for a role in NHEJ.