9756 Nucleic Acids Research, 2012, Vol. 40, No. 19 Figure 4. AP-endonuclease activity of the HhH-motif mutants. The assay was carried out as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 34mer double stranded oligonucleotide containing in the 50-labelled strand an internal THF (H) group, with 64 nM of either the wild-type or the speciﬁed mutant PolXBs in the presence of 1 mM of MnCl2 for the indicated period at 30 C. Figure 5. (A) Potential interaction between PolXBs residue Lys192 and the incoming nucleotide. PolXBs was modelled using as template, the solved structure of PolXDr apoenzyme (PDB 2W9M) (35). Palm subdomains of modelled PolXBs and Polb ternary complex (PDB 1BPY) (39) were further structurally aligned. (B) Binding of mutant polymerases to a gapped DNA. The experiment was carried out as described in ‘Materials and Methods’ section. Five nanomolar of the 2-nt gapped DNA was incubated 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. (C) Gap-ﬁlling reaction of mutants at residue Lys192 on a 2-nt gapped DNA. The assay was carried out as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 2-nt gapped DNA with 62 nM of either the wild-type or the indicated mutant PolXBs in the presence of 8 mM of MgCl2 and 1 mM of dNTPs for the indicated period at 30 C. The unit length of the primer molecule is indicated. (D) Gap-ﬁlling reaction of mutants at residue Lys192 on a 5-nt gapped DNA. The assay was performed essentially as described in (C), using as substrate 1.5 nM of a 5-nt gapped DNA (‘Materials and Methods’).