Caracterización bioquímica de la DNA polimerasa X de bacilus subtilis
- Autores: Benito Baños Piñero
- Directores de la Tesis: José Miguel de Vega (dir. tes.)
- Lectura: En la Universidad Autónoma de Madrid ( España ) en 2011
- Idioma: español
- Tribunal Calificador de la Tesis: José Fernández Piqueras (presid.), Juan Méndez Zunzunegui (secret.), Enrique Viguera Mínguez (voc.), Luis Blanco Dávila (voc.), Antonio Bernad Miana (voc.)
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- Resumen
Bacillus subtilis gene yshC encodes a 64-kDa family X DNA polymerase (PolXBs). Biochemical analysis of the purified enzyme indicates that PolXBs is a strictly template directed DNA polymerase, preferentially acting on DNA structures containing gaps from one to a few nucleotides and bearing a phosphate group at the 5¿ end of the downstream DNA. In addition to the polymerization activity, PolXBs possesses an intrinsic 3¿-5¿ exonuclease activity specialized in resecting the unannealed 3¿-termini in a gapped DNA substrate. Analysis of a PolXBs deletion mutant lacking the C terminal polymerase histidinol phosphatase domain (PHP), present in most of the bacterial/archaeal PolX, as well as of this separately expressed protein region, allows us to state that the 3¿-5¿ exonuclease activity of PolXBs resides in its PHP domain. Furthermore, site directed mutagenesis of PolXBs residues H339, H341, D346, H371, E410, H437, H465, D526 and H528, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. These results led us to propose a potential role for PolXBs in DNA repair.
PolXBs, along with polymerization and 3¿-5¿ exonuclease activities, possesses an intrinsic AP-endonuclease activity. Both, AP-endonuclease and 3¿-5¿ exonuclease activities are genetically linked and governed by the same metal ligands located at the PHP domain. The different catalytic functions of PolXBs enable it to perform recognition and incision at an AP site and further restoration of the original nucleotide in a standalone AP-endonuclease-independent way.
Family X DNA polymerases posses two conserved HhH (Helix-hairpin-Helix) motifs involved in DNA binding. However, bacterial and archaeal PolX have an specific third HhH motif located at the fingers subdomain. Mutational analysis of G130, G132 and K134 residues from this motif in PolXBs demonstrates a role for these residues in DNA interaction, polymerization and exonuclease activities on gapped substrates, allowing us to conclude an essential function for this specific HhH motif in DNA binding as well as a potential role in the proper coordination between the synthetic and degradative activities in bacterial and archaeal family X DNA polymerases.
Many bacterial PolX have a lysine residue preceding the first two catalytic aspartates involved in metal coordination during the polymerization reaction. Substitutions of residue K192 of PolXBs by conservative or non-conservative residues endow the polymerase with a strand displacement ability and improved polymerization activity. These results, together with the decrease in the 3¿-5¿ exonuclease activity observed in the mutant enzymes, led us to suggest a role for this residue in stabilization of the 3¿ terminus in the polymerization active site.
By size exclusion chromatography techniques, PolXBs was observed to form tetramers in vitro, being the PHP domain mainly responsible for this phenomenon. PolXBs binds specifically and with high stability to Holyday junction structures, intermediates of homologous recombination repair pathways. Therefore, it is tempting to speculate that PolXBs tetramers could bind to the four-way Holliday junction structure, suggesting a potential role for this polymerase in homologous recombination in B. subtilis.
