Nucleic Acids Research, 2012, Vol. 40, No. 19 9751 Figure 1. Multiple amino acid sequences alignment of the ﬁngers subdomain and the DxD motif of the palm subdomain of bacterial/archaeal PolXs. The common HhH motif shared by most of PolX members and the one speciﬁcally present in the bacterial/archaeal members are drawn. Numbers between slashes indicate the amino acid position relative to the N-terminus of each DNA polymerase. Numbers in parentheses indicate the length of the intervening amino acid sequence. Because of the large number of sequences, only selected representatives from the Eubacteria and Archea genus are aligned. Names of organisms are abbreviated as follows: Bsub, Bacillus subtilis (GenBank accession number NP_390737); Lmon, Lysteria monocytogenes (GenBank accession number YP_013839); Ssap, Staphylococcus saprolyticus (GenBank accession number YP_301742); Saur, Staphilococcus aureus (GenBank accession number YP_001246578); Dred, Desulfotomaculum reducens (GenBank accession number YP_001112987); Aaeo, Aquifex aeolicus (GenBank accession number NP_213981); Tthe, Thermus thermophilus (GenBank accession number YP_144416); Tden, Thiobacillus denitriﬁcans (GenBank accession number AAZ97399); Mmaz, Methanosarcina mazei (GenBank accession number NP_633918); Faci, Ferroplasma acidarmanus (GenBank accession number ZP_01709777); Mthe, Methanothermobacter thermautotrophicus (GenBank accession number NP_275693); Dred, Deinococcus radiodurans (GenBank accession number NP_294190); Tvol, Thermoplasma volcanium (GenBank accession number NP_111375) and Taqu, Thermus aquaticus (GenBank accession number BAA13425). Conserved residues ( 80% of the aligned polymerases) are indicated with red letters. Conserved glysine and lysine residues of the speciﬁc HhH motif and the lysine residue preceding the ﬁrst catalytic aspartic acid residue are indicated in white letters over a black background. Alignment was made by using the Multalin tool (http:// multalin.toulouse.inra.fr/multalin/multalin.html) and was further adjusted by hand. are stretches of 20 amino acids that bind to the DNA in a sequence independent manner; they are abundant in the prokaryotic and eukaryotic enzymes involved in DNA transactions, such as replication and repair processes (23). They are formed by highly conserved hydrophobic residues organized in two helices of deﬁned length linked by a type-II b-hairpin. Hydrophobic residues form a contact surface between the two helices stabilizing the structure of the motif. The b-hairpin contains two glycine residues separated by a hydrophobic residue. The ﬁrst glycine might be important in the hairpin formation, whereas the second one could provide a contact surface for DNA binding (23). Structural studies performed with Polb bound to a nicked DNA showed that the 8-kDa and ﬁngers HhH motifs interact with the DNA sugar–phos- phate backbone of the downstream and upstream strands, respectively (12,24,25). Such interactions make the DNA to adopt a sharply bent conformation to expose the nascent base pair, allowing the right positioning of the 50-phosphate and 30-OH free ends at their respective active sites, 50-deoxyribose 50-phosphate lyase and poly- merase. Besides the two universal HhH motifs described previously, bacterial and archaeal PolXs have a speciﬁc- ally conserved third HhH motif in the ﬁngers subdomain (26) located three residues C-terminally from the common one (see Figure 1), and whose potential role in DNA binding has not been demonstrated so far. The palm subdomain of PolXs possesses the universal three catalytic aspartates that coordinate the divalent metal cations responsible for the nucleotidyl transfer reaction by the two metal ion mechanism. The ﬁrst catalytic aspartate is preceded by a small residue (glycine) in eukaryotic Polb and Pol (27) that contacts the g-phosphate of the incoming nucleotide (12,13,28,29). However, Polm and TdT, endowed with a terminal trans- ferase activity, contain a histidine residue instead (30). Crystallographic studies have shown that this histidine residue is positioned in such a way that it could form a hydrogen bond with the phosphate of the primer-terminal residue and the g-phosphate of the incoming deoxyribo- nucleoside triphosphate (dNTP), enabling the nucleotidyl transfer reaction in the absence of a template nucleotide (31). Some bacterial PolXs have a moderately conserved lysine residue in that position (26) (see Figure 1). Bacillus subtilis PolX (PolXBs) is a prototype of the bacterial/archaeal subgroup of PolXs that is potentially involved in base excision replair, as it is a strictly DNA-directed DNA polymerase, it is adapted to ﬁll small gaps, allowing ﬁnal sealing of the resulting nick by a DNA ligase (26). Additionally, like in most bacterial/ archaeal PolXs, it has a C-terminal PHP domain (32) including highly conserved residues responsible for an Mn2+-dependent 30-50 exonuclease activity that is able to process unannealed 30 termini (17) and an AP- endonuclease that confers the in vitro ability to recognize, incise and repair AP sites on the polymerase (18). The aim of this work was to ascertain the role of the speciﬁcally conserved third HhH motif placed at the ﬁngers subdomain of bacterial/archaeal PolXs, and the function of the lysine residue that precedes the ﬁrst catalytic aspartate, using PolXBs as a model. The biochem- ical analyses of PolXBs mutants at residues Gly130,