Nucleic Acids Research, 2012, Vol. 40, No. 19 9755 Figure 3. 30-50 exonuclease activity of the HhH motif mutants. (A) Exonuclease activity on ssDNA. The assay was performed as described in ‘Materials and Methods’ section, incubating 1.5 nM of the 15mer oligonucleotide with 125 nM of either the wild-type or the indicated mutant PolXBs in the presence of 1 mM of MnCl2 for the indicated period at 30 C. The unit length of the DNA molecule is indicated. (B) Exonuclease activity on 2-nt gapped DNA. The assay was performed as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 2-nt gapped DNA with 32 nM of either the wild-type or the indicated mutant PolXBs in the presence of 1 mM of MnCl2 for the indicated period at 30 C. (C) Exonuclease activity on 30-mismatched gapped substrates. The assay was carried out as described in (B). AP-endonuclease activity similar to that of the wild-type enzyme, with the only exception of the double mutant G130V/G132V (see Supplementary Figure S2), strongly supporting that the defective phenotypes observed with mutant polymerases G130V and G132V is not because of a general misfolding of the protein. The poor efﬁciency observed with mutant G130V/G132V in all the assayed activities does not allow us to rule out a distortion of the overall structure in this case. Several bacterial PolXs have a lysine residue preceding the catalytic aspartates As described in the ‘Introduction’, eukaryotic Polb and Pol have a glycine residue preceding the ﬁrst catalytic aspartate that interacts with the g-phosphate of an incoming nucleotide, this residue being a histidine respon- sible for the terminal transferase activity of Polm and TdT (31,38). Bacterial PolXs have a moderately conserved lysine/arginine residue in that position (Lys192 in PolXBs; see alignment in Figure 1). As there was not any structurally solved ternary complex of a bacterial/ archaeal PolX when this work started, the palm subdomain of PolXBs was initially modelled on the Polb ternary complex structure (39) (Figure 5A) showing a po- tential interaction between the incoming nucleotide and Lys192 of PolXBs, which precedes the catalytic Asp193 (26). To obtain further details about the role of this amino acid in the activity of PolXBs, the corresponding Lys192 was substituted either by a conservative (K192R) or non-conservative (K192A) residue. These mutants were overproduced and puriﬁed as described in ‘Materials and Methods’ section. Lys192 is not involved in DNA binding The ability of PolXBs mutants to bind a 2-nt gapped sub- strate was evaluated as described previously (‘Materials and Methods’ section). Figure 5B shows that removal of the positive charge in mutant K192A has no effect in PolXBs-DNA complex stability, ruling out a role for this residue in DNA binding. In contrast, substitution of