title: DNA stabilization at the Bacillus subtilis PolX core--a binding model to coordinate polymerase, AP-endonuclease and 3'-5' exonuclease activities.
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.
authors: Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
authors: Benito
authors: B
authors: Baños
authors: Laurentino
authors: L
authors: Villar
authors: Margarita
authors: M
authors: Salas
authors: Miguel
authors: M
authors: de Vega
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology; D001224:Aspartic Acid / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D001224:Aspartic Acid; D004247:DNA; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008239:Lysine