4766 Nucleic Acids Research, 2005, Vol. 33, No. 15 Mg2+ (2 · 10 5 pmol/min ng) or Mn2+ (9 · 10 6 pmol/min ng) as activating divalent metal ions. As a control of speciﬁcity, we carried out a parallel puriﬁcation of a catalytically inactive mutant (see Materials and Methods). In this case, no DNA polymerization activity was detectable in the ﬁnal fraction (data not shown). Therefore, SPAC2F7.06c codiﬁes for a DNA polymerase that we refer to as SpPol4. SpPol4 is a distributive polymerase that lacks 30! 50 exonuclease activity Processivity is a common feature of DNA polymerases involved in extensive DNA synthesis (i.e. replicative poly- merases), and relies on a tight DNA binding and an efﬁcient nucleotide insertion. Conversely, DNA repair enzymes fre- quently display weaker DNA interactions and incorporate nucleotides more slowly and consequently synthesize DNA in a distributive mode. Distributive polymerization is a com- mon feature of all DNA polymerases from the X family (16,25,35,36,45,46). We assessed SpPol4 processivity on a DNA template/primer substrate by analyzing the chain length distribution at several enzyme/DNA substrate ratios. As shown in Supplementary Figure 1, the length of the elongated primer decreased with the enzyme/DNA substrate ratio in agreement with a fully distributive polymerization pattern. This distribu- tive behavior of SpPol4 is also maintained using Mn2+ as metal activator (data not shown). Therefore, we conclude that SpPol4 is a distributive polymerase suited for short-stretch DNA synthesis. Another distinctive feature of replicative DNA polymerases is its proofreading 30!50 exonuclease activity. Three con- served amino acid motifs, named Exo I, Exo II and Exo III, are responsible for the 30!50 exonuclease active site of all proofreading DNA polymerases (47). However, these motifs are absent in the DNA polymerases from the X family includ- ing SpPol4, suggesting that, as other PolX enzymes, SpPol4 has no proofreading activity. We tested this prediction using either a single-stranded oligonucleotide or a template/primer as substrates for 30!50 exonucleolysis. Puriﬁed SpPol4 failed to display any nucleolytic activity on both substrates after 15 min at 30 C (data not shown). This result demonstrates that SpPol4 does not possess 30!50 proofreading activity. SpPol4 prefers small gaps with a 50-phosphate group To further characterize the DNA polymerization activity pre- sent in the puriﬁed SpPol4 fraction, we tested different in vitro assay conditions using deﬁned templated-DNA molecules in the presence of Mg2+ as a cofactor. The puriﬁed protein was able to catalyze dNTP incorporation very efﬁciently either in a template/primer (data not shown) or in the 1 nt gapped sub- strates in a dNTP dosage-dependent manner (Figure 1A and B). However, a signiﬁcant increase (10-fold as an average) in the polymerization capacity was observed when a phosphate group was present at the 50-side of the gap compared with the same gapped DNA molecule having a hydroxyl group at the 50 end of the gap (Figure 1B). Therefore, the DNA sub- strate preference of SpPol4, small gaps with a 50-phosphate group, is compatible with a role in DNA repair. In Polb and Poll a 50-phosphate-dependent increase in pro- cessivity is structurally and functionally related to the presence of the N-terminal 8 kDa domain (5,7,9,48). Since SpPol4 also Figure 1. Gap-filling synthesis and substrate preferences of SpPol4. (A) Scheme of the two types of DNA molecules used: 1 nt gap (GAP1-OH) and 1 nt gap with a 50-phosphate (GAP1-P). Labeled primers (asterisk) and the 50 end hydroxyl (OH) or phosphate (P) groups are indicated. The oligonucleotides used were T13(C)/SP1C/DG1. (B) Gap-filling DNA synthesis of SpPol4 on the 1 nt gapped structures described above. Reactions were carried out as described in Materials and Methods using 125 nM SpPol4 and the indicated concentra- tions of dGTP. Primer extension was analyzed by 8 M urea–PAGE and auto- radiography. Mobility of the unextended primer (P) and the 1 nt (+1) extended primers are indicated at the autoradiograph. (C) DNA-binding capacity of SpPol4 to the 1 nt gapped molecules. EMSA was performed as described in Materials and Methods using none (lanes 1 and 5), 250 nM (lanes 2 and 6), 600 nM (lanes 3 and 7) and 1.2 mM (lanes 4 and 8) SpPol4. Formation of SpPol4:DNA complexes was resolved by native gel electrophoresis on a 4% polyacrylamide gel (80:1; monomer/bis). Mobility of the free DNA (F) and the SpPol4/DNA complexes (C) are indicated at the autoradiograph. (D) Competi- tion analysis of SpPol4 bound to the GAP1-P. EMSA was performed as de- scribed in Materials and Methods, but using the indicated amounts of either 1 nt gapped 50-phosphate (G1-P) or 50-hydroxyl (G1-OH) as unlabeled competitor DNA. The plotted values represent the percentage of labeled GAP1-P that remains bound to SpPol4 after competition, and are the mean of four indepen- dent experiments. contains an N-terminal 8 kDa domain, and is stimulated by the presence of a 50-phosphate group in the 1 nt gapped substrate, we tested if this stimulation was primarily due to differences in the DNA-binding capacity, a step preceding dNTP binding