9754 Nucleic Acids Research, 2012, Vol. 40, No. 19 Figure 2. (A) Binding of the HhH motif mutants to a gapped DNA. The experiment was carried out as described in ‘Materials and Methods’ section, incubating 5 nM of 2-nt gapped DNA with 1 mM of either the wild-type or the indicated mutant PolXBs for 10 minutes at 4 C. The bars chart is the result of three independent experiments. (B) DNA polymerization activity on gapped DNA of HhH motif mutants. The assay was carried out as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 2-nt gapped DNA with 125 nM of either the wild-type or the indicated mutant PolXBs in the presence of 8 mM of MgCl2 and 50 mM of dNTPs at 30 C. The positions corresponding to the non-extended primer and to the ﬁlling-in products are indicated. on ssDNA (Figure 3A) and a gapped DNA (Figure 3B) substrate. Thus, the results would suggest that these residues contact/stabilize the DNA substrate also when the 30 terminus has to be allocated in the catalytic site of the structurally independent PHP domain. Interestingly, mutants at residue Lys134 displayed an activity opposed to their DNA-binding and polymerization capacities. Thus, whereas mutant K134R exhibited a slightly reduced exonuclease activity in comparison with the wild-type PolXBs, mutant K134A showed an exonucleolytic efﬁciency either higher than or similar to that of the wild-type enzyme when acting on ssDNA (Figure 3A) and gapped substrates (Figure 3B), respectively. As aforementioned, previous studies showed that the 30-50 exonuclease activity preferentially degrades 30-ﬂap substrates (17). As it can be seen in Figure 3C, the wild-type enzyme efﬁciently removes the mispaired nucleotides, and the exonucleolytic rate slows down once it reaches the paired dsDNA region, as previously reported (17). On this substrate, mutants G132V and G130V/G132V did not render degradation products. The exonucleolytic activity shown by mutants G130V and K134R was also reduced on this DNA molecule, especially when the double-stranded region was reached. In contrast, mutant K134A presented an extremely high efﬁciency in degrading this kind of substrate (Figure 3C). Recently, PolXBs has been demonstrated to be also endowed with an intrinsic AP-endonuclease activity at its PHP domain, sharing the same catalytic residues re- sponsible for the 30-50 exonuclease activity (18). This activity qualiﬁes PolXBs to perform in vitro recognition and incision at an AP site and further restoration of the original nucleotide in a stand-alone AP endonuclease in- dependent way. To study the involvement of the HhH motif in aiding the AP endonuclease reaction, mutant polymerases were incubated in the presence of a DNA containing an internal THF (a stable analogue that mimics an abasic site) at the 19 position (see ‘Materials and Methods’ section). As observed in Figure 4, the wild-type enzyme promoted cleavage at the 50 side of the THF position, rendering the expected 18mer product. The shorter bands observed are caused by the action of the 30- 50 exonuclease activity on the 30-end resulting after incision at the AP site, as previously described (18). As it is shown, mutations introduced in residues of the HhH motif impaired either severely (G130V, G132V and G130V/G1232V) or moderately (K134R and K134A) the AP-endonuclease activity of PolXBs, leading to conclude the importance of this HhH motif also in the stabilization of AP-bearing DNA substrates. We have previously shown that PolXBs can also intro- duce an internal nick on ssDNA substrates containing a THF much more efﬁciently than on dsDNA (18). On this substrate, mutant polymerases exhibited an