Table 1. Incoming nucleotide afﬁnity displayed by PolXBs and mutant derivatives K192R and K192A PolXBs Km (mM) Wild-type 10.3 ± 1.75 K192R 13.9 ± 1.9 K192A 15 ± 3.9 Kinetic parameter was calculated from primer extension experiments, as described under ‘Materials and Methods’ section. Km stands for the Michaelis–Menten constant. Errors are calculated as mean values. lysine by the positively charged residue arginine provided mutant K192R with a DNA binding efﬁciency two-fold higher than that of the wild-type PolXBs. Mutant K192R shows a strand displacement phenotype Figure 5C shows the ability to ﬁll the 2-nt gapped sub- strate by mutants at residue Lys192. As it can be observed, the lack of the positively charged side chain in mutant K192A greatly impaired the gap-ﬁlling reaction, despite its wild-type DNA binding ability (aforementioned). Interestingly, although mutant K192R ﬁlled the gap nearly as efﬁciently as the wild-type enzyme, DNA syn- thesis was not restricted to the completion of the gap, as faint bands corresponding to longer products could be observed (18–19mer bands in Figure 5C), suggesting the acquisition of a partial strand displacement capacity. Similar assays were also conducted on a 5-nt gapped DNA. As shown in Figure 5D, wild-type PolXBs ﬁlled the gap to completion, synthesis being restricted to the ﬁve nucleotides of the gap (20mer band). The speciﬁc blockage at the +1 position was previously described to be independent of the template sequence and length of the gap (26). It has been described in other DNA polymerases that their 30-50 exonuclease activity prevents strand dis- placement synthesis, such as Pold during Okazaki fragment maturation (40). To analyse whether the absence of strand displacement synthesis by PolXBs is determined by its intrinsic 30-50 exonuclease activity, the gap ﬁlling activity of the exonuclease deﬁcient PolXBs de- rivatives D346A and H371A (18) was also analysed. As shown in Figure 5D, mutant enzymes displayed a gap ﬁlling efﬁciency similar (H371A) or two-fold higher (D346A) than that exhibited by the wild-type enzyme, the longest elongation product being mainly the 20mer oligonucleotide. This result means that, at least under these assay conditions, exonuclease activity is not respon- sible for preventing strand displacement synthesis by PolXBs. Again, mutant K192R carried out proﬁcient gap-ﬁlling reaction coupled to a partial downstream strand displacement, as deduced from the yield of 21– 22mer products (Figure 5D). In contrast, mutant K192A had an impaired polymerization activity. Measurement of the Km value (in mM) for the incoming nucleotide showed no substantial differences among the wild-type and the two mutant polymerases K192R and K192A (see Table 1). This result and the nearly wild-type DNA binding capability of mutant K192A would suggest that its poor polymerization capacity could be because of a Nucleic Acids Research, 2012, Vol. 40, No. 19 9757 Figure 6. PHP-dependent activities of mutants at PolXBs residue Lys192. (A) 30-50 exonuclease activity. The assay was performed as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 2-nt gapped structure 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. (B) AP-endonuclease activity. The assay was carried out as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 34mer double stranded oligonucleotide containing an internal THF group with 64 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. non-functional orientation of the primer-terminus at the polymerisation active site. Changes introduced at Lys192 residue reduced the 30-50 exonuclease activity on gapped molecules (Figure 6A) and the AP-endonuclease one (Figure 6B). These results seem to indicate that this residue might be involved in orienting/ stabilizing the DNA substrate also at the PHP active site. DISCUSSION PolXBs exhibits the general enzymatic features of the PolX members: dependence on divalent metal ions, re- quirement of a template strand to direct DNA synthesis, a distributive polymerization pattern, preferential use of gapped DNA structures bearing a downstream 50-phosphate group and favourable insertion of the com- plementary deoxyribonucleoside monophosphate (26). This polymerase shares with most of bacterial/archaeal