molecule speciﬁed in each case and the indicated amount of the wild-type or mutant PolXBs. After incubation for the indicated period at 30 C, reactions were stopped by adding 10 mM EDTA and were analysed by 8 -M urea-20% PAGE and autoradiography. AP-endonuclease activity assays The 12.5 ml of incubation mixture contained 50 mM Tris-HCl, pH 7.5, 1 mM MnCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 1.5 nM of 50-labelled hybrid THF/THFc and 64 nM of the wild-type or mutant PolXBs. Samples were incubated at 30 C for the indicated period and stopped by adding 10 mM of EDTA. Reactions were analysed by 8 -M urea-20% PAGE and autoradiography. RESULTS Bacterial and archaeal PolXs possess a speciﬁc HhH motif Figure 1 shows a sequence alignment of the well-delimited ﬁngers subdomain of several representatives of bacterial/ archaeal PolXs. This subdomain speciﬁcally contains, along with the common HhH motif that, by analogy with Polb, would interact with the upstream primer strand of a gapped DNA (12), another HhH motif placed three amino acids C-terminally apart (26). Besides the two conserved glycine residues predicted to be critical for DNA–protein recognition, this motif contains a conserved lysine residue two nucleotides apart from the second glycine, as described for other HhH motifs (23). Crystallographic resolution of the structure of Deinococcus radiodurans PolX apoenzyme (PolXDr) showed the corresponding HhH motif formed by helices H and I of the ﬁngers subdomain, the latter providing the link with the catalytic palm subdomain (35). The relative orientation of both subdomains compels PolXDr to adopt a non-standard extended conformation that makes a po- tential DNA binding role unpredictable for this HhH motif. To ascertain the role of this motif of bacterial/archaeal PolXs, we introduced the aliphatic chain valine in lieu of the corresponding PolXBs residues Gly130 and Gly132 to obtain the mutants G130V, G132V and G130V/G132V, as such change has been previously reported in mutational studies carried out at the glycine residues of other HhH motifs (36,37), and because a valine residue is not found in the HhH motifs aligned in Doherty et al. (23). On the other hand, Lys134 was substituted by either a conserva- tive (K134R) or non-conservative (K134A) residue. All these mutant polymerases were overexpressed and puriﬁed as described in ‘Materials and Methods’ section, and their catalytic activities were analysed by in vitro bio- chemical assays. The HhH motif is essential for DNA binding As most reported members of the PolX family, PolXBs acts preferentially on gapped DNA molecules (17,26). Because of the inefﬁciency shown by this polymerase to Nucleic Acids Research, 2012, Vol. 40, No. 19 9753 give rise to a stable retardation band in electrophoretic mobility shift assays (not shown), analysis of the efﬁciency of the wild-type and mutant derivatives to bind a 2-nt gapped DNA molecule was performed by nitrocellulose ﬁlter-binding assays (see ‘Materials and Methods’ section). As shown in Figure 2A, substitution of glycine residues in mutants G130V, G132V and G130V/G132V greatly impaired the binding capability of the polymerase, their efﬁciency being 10-fold lower than that displayed by the wild-type enzyme. Additionally, the presence of a positive charge in position 134 seems to be also important to maintain the stability of the PolXBs–DNA complex, as its removal in mutant K134A diminished DNA binding efﬁciency 2.7-fold with respect to the wild-type polymer- ase. Contrarily, mutant K134R showed an improved binding ability. Altogether, these results indicate a DNA-binding role for the speciﬁcally conserved HhH motif at the ﬁngers subdomain of bacterial/archaeal PolXs. Mutant polymerases at the HhH motif have an altered polymerization activity on gapped DNA PolXBs has been proposed to be involved in the repair of DNA damages because of its intrinsic ability to accommo- date itself in gaps of one to few nucleotides, intermediates that arise during DNA repair processes (26). Figure 2B shows the effect of the mutations in ﬁlling a 2-nt gapped DNA substrate (see ‘Materials and Methods’ section). Whereas the wild-type PolXBs elongates about 50% of the initial substrate at the shortest elongation time assayed, ﬁlling the gap 1–2 minutes after starting the reaction, mutants G132V and G130V/G132V did not show any noticeable polymerization activity in the condi- tions assayed, mutant G130V giving rise only to a +1 faint elongation band that is 12% of the wild-type activity, in good correlation with their impaired DNA binding (afore- mentioned). Accordingly, the increased binding capability of mutant K134R (aforementioned) could be responsible for its higher gap-ﬁlling proﬁciency that enables the enzyme to synthesize the +2 product at the shortest elong- ation time assayed (see Figure 2B). Conversely, the dimin- ished DNA binding capability of mutant K134A would account for its 2-fold lower polymerization activity than that of the wild-type polymerase. The proportional increase in the polymerization reaction relative to the amount of DNA observed with mutants G130V and G132V is consistent with a defect in DNA binding as being responsible for the reduced poly- merization activity (see Supplementary Figure S1). Mutations introduced at the HhH motif affect the PHP-dependent activities of PolXBs PolXBs has an Mn2+-dependent 30-50 exonuclease activity at its C-terminal PHP domain that capacitates the enzyme to resect mismatched 30-termini in gapped DNA substrates (17). To ﬁnd out whether the HhH residues under study could assist this nucleolytic activity, mutant polymerases underwent 30-50 exonuclease assays (see ‘Materials and Methods’ section). Unexpectedly, as shown in Figure 3, mutations introduced at residues Gly130 and Gly132 impaired the exonuclease activity of PolXBs when acting