9760 Nucleic Acids Research, 2012, Vol. 40, No. 19 Figure 8. (A) Model of the PHP motion. The PHP and polymerization subdomains are coloured as in Figure 7A. Catalytic residues of the PHP domain are represented as orange sticks. The 30 terminal nucleotide of the upstream primer strand is represented as spheres. Curved arrow indicates the proposed movement of the PHP domain. See main text for details. (B) Electrostatic surface of the modelled PHP domain of PolXBs. Superﬁcial placement of the catalytic active site is indicated. (C) Scheme depicting the proposed PHP motion during the 30-50 exonucleolytic removal of mispaired 30 termini and the repair of AP sites by PolXBs. PolX core and PHP domains are represented as green and cyan boxes, respectively. See main text for details. (D) Modelling of the PolXBs residue Lys192 at the polymerization active site. Incoming nucleotide, 30 primer-terminus and Lys192 are represented as yellow, violet and cyan spheres. Figure was made using PyMOL software (http://www.pymol.org). presented here rule out a role of this residue in general stabilization of the PolX–DNA complex, as the K192A mutant did not show a reduction in its DNA binding cap- ability. However, in this case, the lack of the lysine side chain made the polymerase deﬁcient in elongating the primer strand. Its nearly wild-type Km value for the incoming nucleotide suggests a non-suitable orientation and/or stabilization of the primer-terminus at the polymerization site as the most likely reason for its deﬁ- cient polymerization activity. This hypothesis was initially substantiated by the PolXDr structure in which the cor- responding Arg196 was postulated to stabilize the primer-terminus at the polymerization site through con- tacting with the 30-phosphate, taking over the role of residues Arg254, Arg488 and Lys616 of Pol , Polb and PolIII, respectively (35). Additionally, further modelling of PolXBs residue Lys192 on the recently solved ttPolX ternary complex (which has a glycine residue instead) shows the side chain positioned just in between the primer-terminal residue and the incoming nucleotide (see Figure 8D), in a similar fashion to His229 in Polm (31). It is noteworthy that in the absence of incoming nucleotide, mutant K192A also displayed a reduced 30-50 exonuclease and AP endonuclease activities, maybe because of the aforementioned distortion that would make the right al- location difﬁcult either for the upstream 30 terminus or for the AP site at the PHP catalytic centre, suggesting a par- ticipation of this residue in the coordination of the