9758 Nucleic Acids Research, 2012, Vol. 40, No. 19 PolXs a C-terminal PHP domain. In PolXBs, this domain is endowed with an inherent 30-50 exonuclease activity, de- pendent on highly conserved residues, and that enables the enzyme to process unannealed 30-termini and that made us to suggest the presence of this activity in the rest of PHP-containing bacterial/archaeal PolXs (17). In addition, PolXBs possesses also an intrinsic AP-endonuclease activity genetically linked to the exonucleolytic one and governed by the same metal ligands of the PHP domain (18). Altogether, these three different catalytic functions, polymerization, 30-50 exonucleolysis and AP-endonucleolysis capacitate PolXBs to perform, at least in vitro, recognition and incision at an AP site and further repair the original nu- cleotide in a stand-alone AP-endonuclease independent way (18). Preferential binding of PolXs to gapped substrates largely relies on two universally present HhH motifs, one at the N-terminal 8-kDa domain and the other at the ﬁngers subdomain (12,25,41,42). Crystallographic resolution of Polb complexes has shown the backbone of the downstream DNA of the gap interacting with the protein through the HhH motif of the 8-kDa domain, whereas that of the ﬁngers contacts with the sugar–phos- phate backbone of the upstream primer strand stabilizing it at the polymerization active site (6,12,13,24,25,41). In addition, bacterial/archaeal PolX members also contain a third putative HhH motif whose function remained to be determined. When this work started, PolXDr apoenzyme was the only bacterial PolX member whose structure had been solved (35). In those crystals the PolX core of the polymerase exhibited a stretched out conformation instead of the commonly found closed right hand, the ﬁngers subdomain being in a totally rotated position relative to the palm subdomain compared with other family X DNA polymerases (35). This fact made it difﬁ- cult to envision a DNA-binding role for the speciﬁcally conserved HhH motif of the ﬁngers subdomain of bacter- ial/archaeal PolXs. The results presented here underline the importance of the glycine residues of the latter HhH motif to stabilize the binding of PolXBs to a gapped substrate and to an AP-containing DNA, allowing correct location of the primer-terminus at the polymerization and PHP active sites. In addition, the results obtained with mutants at residue Lys134 are also in accordance with a DNA binding role for this motif. Thus, mutant K134R showed an improved DNA binding capability that resulted in enhanced polymerization efﬁciency, although the exo- nuclease and AP- endonuclease activities were reduced in comparison to those of the wild-type enzyme, suggesting that the presence of an arginine residue in that position stabilizes the primer-terminus in a conformation prone to elongation. In this sense, the decreased DNA binding cap- ability of mutant K134A caused a diminished polymerization activity. Here, the poorer stabilization of the primer strand at the polymerization site could be re- sponsible for the enhanced 30-50 exonuclease observed, mainly on mispaired 30 termini. Propensity of a lysine residue at this position, previously described for other HhH motifs, suggested that it could interact with DNA–phosphate groups in a similar manner to the same residues in a P-loop structure (23), as demonstrated for Lys68 of the Polb 8-kDa HhH motif that was shown to contact the phosphate backbone of the downstream strand of a gapped molecule (24). Altogether, these results point to a role for the speciﬁcally conserved HhH motif of the ﬁngers subdomain of bacterial/archaeal PolX in general stabilization of DNA substrates and in the co- ordination among the different activities of these enzymes, as well as to suggest a common DNA binding site for the three activities of these polymerases. Considering PolXBs apoenzyme folded as PolXDr, because of the homology shared between both enzymes, only a pronounced struc- tural reorientation of the ﬁngers subdomain on binding the substrates would account for the results presented here. Recently, the crystallographic structure of ttPolX has been solved, showing that the complex with an incoming nucleotide (binary complex) adopts the same extended structure observed in the PolXDr apoenzyme (33). Comparison of the crystal structures of the ttPolX binary and ternary (with a 1-nt gapped DNA) complexes showed a ﬁne superposition of the palm, thumb and PHP domains, in contrast to the 8-kDa domain and ﬁngers subdomain that are shifted to a great extent to bind the DNA (33). Figure 7A shows a model of the PolXBs struc- ture obtained with the SWISS-MODEL software (43,44), using ttPolX ternary complex as template. PolX core adopts the classical cupped shape observed in Polb on binding DNA wrapping this substrate around. In this con- formation, the HhH motif of bacterial/archaeal PolX ﬁngers is now placed in such a way that the two glycines and the lysine residues would contact the upstream region of the gapped DNA, in good agreement with the DNA binding role for this HhH motif that is proposed here. In addition, the structural analysis of the common and spe- ciﬁcally conserved HhH motifs of the ﬁngers subdomain of ttPolX leads us to conclude that they are integrated as a part of a ﬁve-helical domain termed (HhH)2 (45). This domain classically consists of two consecutive HhH motifs linked by a connector helix, showing a conserved hydrophobic core comprising seven residues (see Figure 7B), one residue from each a-helix and each hairpin (45). The symmetric structure of this (HhH)2 domain would mirror the symmetry of the DNA double helix enabling strong non-speciﬁc binding. As shown in Figure 8A, the modelled PolXBs core is folded in a catalytically competent manner for polymerization in the ternary complex. The PHP domain is attached to the thumb subdomain by a 30 amino acids long linker, its b-barrel conformation forming an almost right angle with the longitudinal axis of the DNA bound to the PolX core. There is not any evident DNA binding cleft in the PHP domain, the catalytic residues being solvent exposed and placed close to the molecular surface of the domain but far away from the DNA bound to the only binding cleft of the polymerase (see Figure 8A). Hence, the relative orientation of the PolX core and the PHP domain could not account either for the appropriate coordination among the catalytic activities of the enzyme shown to occur during the