9752 Nucleic Acids Research, 2012, Vol. 40, No. 19 Gly132 and Lys134 (HhH motif) lead us to deﬁne a general DNA binding role for this motif during polymerization, 30- 50 exonuclease and AP-endonuclease activities. Additionally, in vitro studies of mutants at residue Lys192 (preceding the ﬁrst catalytic aspartate) allow us to predict a function for this lysine in the orientation of the DNA substrates at the polymerization and PHP active sites. Based on the results presented here and in light of the recently solved structure of the bacterial Thermus thermophilus PolX ternary complex (ttPolX) (33), we propose a structural model to account for the coordination of the different catalytic activities of bacterial PolXs. MATERIALS AND METHODS Nucleotides and proteins Unlabelled nucleotides were purchased from GE Healthcare. [g-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer. T4 polynucleotide kinase was purchased from New England Biolabs. Wild-type PolXBs was expressed and puriﬁed as described by Ban˜ os et al. (26). Oligonucleotides Oligonucleotides sp1 (50-GATCACAGTGAGTAC) and sp1+3 (50-GATCACAGTGAGTACCGG) were used as primer strands. Oligonucleotides sp1c+18 (50-ACTGGC CGTCGTTCTATTGTACTCACTGTGATC) and sp1c+15 (50-ACTGGCCGTCGTTTTGTACTCACTGT GATC) that have a 50-terminal extension of 18 and 15 nt, respectively, in addition to the sequence comple- mentary to sp1 and to the 50 15 nt of sp1+3, were used as the template strand. The 50 phosphorylated oligo- nucleotide dws(P) (50-AACGACGGCCAGT), comple- mentary to the last 13 50-nucleotides of sp1c+18 and sp1c+15, was used as a downstream oligonucleotide to construct gapped structures of ﬁve and two nucleotides, respectively. Oligonucleotides sp1 and sp1+3 were 50-labelled using [g-32P]ATP (3000 Ci/mmol) and T4 poly- nucleotide kinase. To analyse the DNA-dependent polymerization activity of the protein on different DNA- gapped structures and the 30-50 exonuclease activity, the labelled primers and the downstream dws(P) oligonucleo- tide were hybridized either to the template sp1c+18 (5-nt gap) or to sp1c+15 (2-nt gap). The substrate for the analysis of the AP-endonuclease activity was obtained by hybridizing 32P-labelled 50 oligonucleotide THF (50-G TACCCGGGGATCCGTACHGCGCATCAGCTGCA G), labelled as described previously where H stands for tetrahydrofuran (THF), with the complementary oligo- nucleotide THFc (50-CTGCAGCTGATGCGCAGTAC GGATCCCCGGGTAC). The oligonucleotides were purchased from Invitrogen. All the hybridizations were performed in the presence of 0.2 -M NaCl and 60-mM Tris-HCl, pH 7.5. Site-directed mutagenesis of PolXBs PolXBs mutants G130V, G132V, G130V/G132V, K134R, K134A, K192R and K192A were obtained by using the QuickChange site-directed mutagenesis kit provided by Amersham Pharmacia. Plasmid pET28-PolXBs contain- ing the PolXBs gene was used as a template for the mu- tagenesis reaction. Expression and puriﬁcation of the mutant proteins were performed as described for the wild-type PolXBs (26). DNA binding assay The assay was carried out using 5 nM of a 50-labelled 2-nt gapped DNA as substrate. The substrate was incubated with 1 mM of the wild-type or mutant PolXBs for 10 mi- nutes at 4 C in the presence of 50 mM Tris-HCl, pH 8, 25 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM MnCl2 and was ﬁltered through nitrocellulose ﬁlters as described by Wong and Lohman (34). Radiolabelled DNA retained in each ﬁlter was quantiﬁed using the Cerenkov radiation and was represented as bars after sub- tracting the amount of DNA that remained bound to the ﬁlter in the absence of enzyme (background). DNA polymerization assays on gapped DNA molecules The 12.5 ml of incubation mixture contained 50 mM Tris- HCl, pH 7.5, 8 mM MgCl2, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 1.5 nM of the hybrid DNA indicated in each case and the speciﬁed concentration of polymerase and dNTPs. After incubation for the indicated period at 30 C, reactions were stopped by adding 10 mM of EDTA and analysed using 8 -M urea-20% PAGE and autoradiography. Measurement of the Km value for the incoming nucleotide The incubation mixture contained, in a ﬁnal volume of 12.5 ml, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 4% glycerol, 0.1 mg/ml BSA, 8 mM MgCl2 and 1.5 nM of the 50-labelled 2-nt gapped DNA. Reaction times and enzyme concentration were adjusted for each polymerase (3.75 nM, 6.25 nM and 10 nM of the wild-type, K192R and K192A polymerase, respectively) to optimize product detection while ensuring that all reactions were conducted in the steady-state. Only those reactions that fell within the linear range of substrate utilization (<30% primer extension) were used for analysis. Samples were incubated for 1 minute at 30 C in the presence of increasing concentration of the incoming nucleotide and were quenched by adding 10 mM EDTA. Reactions were analysed by electrophoresis in 8 -M urea-20% PAGE and were quantiﬁed using a Molecular Dynamics PhosphorImager. Formation of the extended product was plotted against dNTP concentration. Apparent value of Michaelis–Menten constant (Km) was obtained by least-squares non-linear regression analysis to a rect- angular hyperbola using KaleidaGraph 3.6.4 software. Data are shown as mean ± SD corresponding to four in- dependent measurements. Exonuclease activity assays on 50-labelled DNA substrates The 12.5 ml of incubation mixture contained 50 mM Tris-HCl, pH 7.5, 1 mM MnCl2, 1 mM DTT, 4% glycerol, 0.1 of mg/ml BSA, 1.5 nM of the DNA