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. 2012 Oct;40(19):9750-62.
doi: 10.1093/nar/gks702. Epub 2012 Jul 25.

DNA Stabilization at the Bacillus Subtilis PolX Core--A Binding Model to Coordinate Polymerase, AP-endonuclease and 3'-5' Exonuclease Activities

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DNA Stabilization at the Bacillus Subtilis PolX Core--A Binding Model to Coordinate Polymerase, AP-endonuclease and 3'-5' Exonuclease Activities

Benito Baños et al. Nucleic Acids Res. .
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Abstract

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3'-5' exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.

Figures

Figure 1.
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.
Figure 2.
Figure 2.
(A) Binding of the HhH motif mutants to a gapped DNA. The experiment was carried out as described in ‘Materials and Methods’ section, incubating 5 nM of 2-nt gapped DNA with 1 µM 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. (B) DNA polymerization activity on gapped DNA of HhH motif mutants. The assay was carried out as described in ‘Materials and Methods’ section, incubating 1.5 nM of a 2-nt gapped DNA with 125 nM of either the wild-type or the indicated mutant PolXBs in the presence of 8 mM of MgCl2 and 50 µM of dNTPs at 30°C. The positions corresponding to the non-extended primer and to the filling-in products are indicated.
Figure 3.
Figure 3.
3′-5′ 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 3′-mismatched gapped substrates. The assay was carried out as described in (B).
Figure 4.
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 5′-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.
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 Polβ 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 µM 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 µM 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’).
Figure 6.
Figure 6.
PHP-dependent activities of mutants at PolXBs residue Lys192. (A) 3′-5′ 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.
Figure 7.
Figure 7.
(A) Ribbon representation of the structural model of PolXBs bound to DNA. Model for PolXBs was provided by the homology-modelling server SWISS-MODEL, using the crystallographic structure of the ternary complex of ttPolX as template (PDB code 3AUO) (33). The PHP domain is shown in light blue, the 8-kDa domain in green, fingers in gold yellow, palm in red and thumb in magenta. Bound DNA is represented as spheres. The upstream, template and downstream strands are coloured in yellow, cyan and dark blue, respectively. Glycines and lysine of the specifically conserved HhH motif are represented as orange and green spheres, respectively. (B) Fingers subdomain of bacterial PolXs contains a (HhH)2 domain. PolXBs structure was modelled as described in (A). Common and specifically conserved HhH motifs are represented as red and cyan ribbons, respectively, whereas connector α-helix is coloured in orange. PolXBs residues forming the conserved hydrophobic core are represented as magenta sticks. Figure was made using PyMOL software (http://www.pymol.org).
Figure 8.
Figure 8.
(A) Model of the PHP motion. The PHP and polymerization subdomains are coloured as in Figure 7A. Catalytic residues of the PHP domain are represented as orange sticks. The 3′ terminal nucleotide of the upstream primer strand is represented as spheres. Curved arrow indicates the proposed movement of the PHP domain. See main text for details. (B) Electrostatic surface of the modelled PHP domain of PolXBs. Superficial placement of the catalytic active site is indicated. (C) Scheme depicting the proposed PHP motion during the 3′-5′ exonucleolytic removal of mispaired 3′ termini and the repair of AP sites by PolXBs. PolX core and PHP domains are represented as green and cyan boxes, respectively. See main text for details. (D) Modelling of the PolXBs residue Lys192 at the polymerization active site. Incoming nucleotide, 3′ primer-terminus and Lys192 are represented as yellow, violet and cyan spheres. Figure was made using PyMOL software (http://www.pymol.org).

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