Nucleic Acids Research, 2012, Vol. 40, No. 19 9759 Figure 7. (A) Ribbon representation of the structural model of PolXBs bound to DNA. Model for PolXBs was provided by the homology-modelling server SWISS-MODEL, using the crystallographic structure of the ternary complex of ttPolX as template (PDB code 3AUO) (33). The PHP domain is shown in light blue, the 8-kDa domain in green, ﬁngers in gold yellow, palm in red and thumb in magenta. Bound DNA is represented as spheres. The upstream, template and downstream strands are coloured in yellow, cyan and dark blue, respectively. Glycines and lysine of the speciﬁcally conserved HhH motif are represented as orange and green spheres, respectively. (B) Fingers subdomain of bacterial PolXs contains a (HhH)2 domain. PolXBs structure was modelled as described in (A). Common and speciﬁcally conserved HhH motifs are represented as red and cyan ribbons, respectively, whereas connector a-helix is coloured in orange. PolXBs residues forming the conserved hydrophobic core are represented as magenta sticks. Figure was made using PyMOL software (http://www.pymol.org). resection of a misaligned 30 terminus (17) and the repair of an abasic site (18) or for the effect that the HhH mutations studied here had on the different activities of PolXBs. Indeed, and as mentioned previously, our results strongly favour a common DNA binding site for the three enzymatic functions of PolXBs. If there is only a DNA binding cleft, and considering the disposal of the domains in the current structures of the homologous ttPolX, the question that arises is how the bound DNA can access to the polymerization and PHP active sites to coordinate polymerization, 30-50 exonuclease and AP-endonuclease activities. If the DNA does not go to the PHP domain, the PHP domain must go to the DNA An appealing hypothesis would be that the incapacity of the DNA polymerase to elongate a primer, caused by the presence of a mismatched 30 terminus at the polymerization active site, the absence of an incoming nu- cleotide or the presence of an AP site could promote a rotation of the PHP domain toward the upper surface (solvent accessible) of the PolX core. Such a rotation could be possible by virtue of the long linker that connects both polymerase portions, allowing the superﬁ- cial PHP active site to reach and either remove a non-extendable 30 terminus or catalyze hydrolysis at an AP site placed at the polymerization active site (see Figure 8A and C). Once the PHP domain goes back to the initial position, the entrance of an incoming nucleotide would promote further elongation of the resulting sanitized 30 ends. The model proposed here could account for the role of the HhH motif in general DNA binding and would explain how the three enzymatic activities of PolXBs can be coordinated during the repair process using a single DNA binding cleft. In addition, it would give an explanation also for the follow- ing: (i) our previous results showing a higher efﬁciency of PolXBs to recognize and bind speciﬁcally and stably to an AP-containing dsDNA molecule, in comparison to a non-damaged dsDNA (18), (ii) the signiﬁcant reduction of the 30-50 exonuclease activity of PolXDr because of mu- tations introduced at the common HhH motif of its ﬁngers subdomain (36) and (iii) the tight interaction observed between the independently expressed polymerization and PHP domains of ttPolX when they were mixed, as the model described here would account for the large interface contact area proposed to exist (19). Although the PHP movement would be the major structural change required, the model demands for other rearrangements in the PolX core to allow the PHP domain to lie over the polymerization active site, mainly in the presence of an AP-containing DNA. In this case, the 8-kDa domain could not be oriented exactly as it is in the reported ternary complexes because a downstream 50-phosphate end would result only after AP endonucleolysis. However, this kind of reorientations could be feasible, as a substantial movement of the 8-kDa and ﬁngers subdomains has been shown to occur on binding DNA in ttPolX owing to their high ﬂexibility (33). Role of PolXBs residue Lys192 As mentioned previously, PolXBs possesses a lysine residue, moderately conserved among bacterial PolXs, preceding the two ﬁrst catalytic aspartates. The results