Polymerases specialized in damage tolerance and DNA double-strand break repair

• Autores: Guillermo Sastre Moreno
• Directores de la Tesis: José F. Ruiz Pérez (dir. tes.), Luis Blanco Dávila (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2016
• Idioma: inglés
• Tribunal Calificador de la Tesis: José Fernández Piqueras (presid.), Antonio Bernad Miana (secret.), Juan Méndez Zunzunegui (voc.), Ian Holt (voc.), Óscar Fernández Capetillo (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • In spite of the fact that all DNA polymerases catalyse the same chemical reaction, they all harbour unique properties that suit them to synthesize DNA as efficiently as possible in the different mechanisms in which they are involved. In this Doctoral Thesis we have studied the human DNA polymerases Polλ and Polμ and their ortholog in yeast Pol4, which are specialized in double-strand break (DSB) repair, and also PrimPol, a human DNA primase/polymerase specialized in damage tolerance. In this work we have attempted to further understand the function of these DNA polymerases, the structural bases of their adaptation to those functions and the regulation of their activity.

    Pol4 is the only member of the family X of DNA polymerases (PolX) found in yeast, and it is named SpPol4 in S. pombe. In this work we show that the low sugar discrimination property of SpPol4 is relevant during the tolerance of the pre-mutagenic lesion 8oxodG. SpPol4 incorporates ATP, the most abundant ribonucleotide, preferably opposite 8oxodG, also during DSB repair. Although this activity leads to the generation of a base pair with both a sugar and base errors, it is not problematic since the ribonucleotide can be later repaired by the ribonucleotide excision repair mechanism, which we show to be coupled to the incorporation of the correct nucleotide, dCTP, opposite 8oxodG.

    Using also yeast as model, we demonstrate that ScPol4, the only PolX in S. cerevisiae, is phosphorylated by the Tel1/ATM kinase in response to DNA damage. This evidence prompted us to evaluate the regulation by phosphorylation of its closest human ortholog, Polλ, and hence, we demonstrate, that Polλ is phosphorylated by DNA-PK, the kinase that regulates the non-homologous end joining pathway (NHEJ), modulating its activity by stimulating the interaction with Ku80 in response to DNA damage. On the other hand, we have also characterized two tumour variants of the other human PolX specialized in NHEJ, Polμ, and we demonstrate that both tumour variants decrease the efficiency and fidelity of Polμ by diminishing its template dependence during NHEJ in vitro.

    Finally, we have gained further insight into the properties of PrimPol by demonstrating that it is endowed with the ability to transpolymerize across discontinuous templates, even when the primer and template have no complementarity. We also show how the metal cofactors Mg2+ and Mn2+ modulate the previously described translesion properties of PrimPol, providing a good balance between fidelity and efficiency, although we suggest that its active site is adapted to use Mn2+ with maximum efficiency.