Bacillus subtilis rada/sms and reca contribute in concert to double-strand break repair and natural transformation, and with disa to dna damage tolerance

• Autores: Rubén Torres Sánchez
• Directores de la Tesis: Juan Carlos Alonso Navarro (dir. tes.), Mario Mencía Caballero (tut. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2019
• Idioma: español
• Tribunal Calificador de la Tesis: José Berenguer Carlos (presid.), José Antonio Tercero Orduña (secret.), Ernesto Arias Palomo (voc.), Patrice Polard (voc.), Andrés Aguilera López (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Química
    • Bioquímica
  • Ciencias de la vida
    • Genética
    • Microbiología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • DNA is constantly subjected to alterations in all living cells. If unrepaired, these alterations can lead to mutations or even cell death, and this is the reason why cells have evolved diverse mechanisms that repair or tolerate the damage to survive in its presence. When cells are actively dividing, one of the major consequences of unrepaired damages is the arrest of the replication fork machinery. Bacillus subtilis RadA/Sms and DisA have been described to collaborate in the mechanisms that cope with this arrest, by coordinating the timing of cell proliferation with the DNA damage response. The mechanisms that act upon replication fork stalling include fork reversion, template switching and translesion synthesis; but when replication forks collapse, RecA-mediated homologous recombination is the preferred pathway. This DNA strand exchange mechanism performed by RecA in DNA repair shares common steps with the one occurring in natural chromosomal transformation, a crucial process for the acquisition of genetic diversity and for the restoration of mutated genes, that plays a central role in the evolution and the spread of pathogenicity traits and antibiotic resistance genes. Since both DNA repair and natural transformation are essential for bacterial survival and have crucial roles in human health, the role of RadA/Sms, DisA and RecA in these processes has been studied in this work.

    RadA/Sms conserved C4 (or Zn finger domain), H1 (or Walker A domain) and KNRFG motifs are crucial for survival upon replication fork arrest and for natural transformation. DisA-mediated synthesis of the essential c-di-AMP messenger and DNA binding are crucial for survival upon replication fork stalling, but DisA is dispensable for natural transformation. RadA/Sms and its mutants in the conserved C4, H1 and KNRFG motifs bind preferentially ssDNA and HJ DNA. RadA/Sms shows ATPase activity that cannot be further stimulated by DNA substrates, while the interaction of RadA/Sms C4 motif mutants with DNA stimulates their ATPase activity. RadA/Sms and its mutant variants in the C4 motif unwind DNA in the 5´3´ direction. RecA interacts with wt RadA/Sms, but not with RadA/Sms C4 mutants, and loads it on the DNA to promote unwinding of non-cognate substrates. This interaction is crucial to recruit RadA/Sms onto displacement loop (D-loop) DNA, and both proteins in concert facilitate D-loop extension and integration of ssDNA during chromosomal transformation. During double-strand break repair, RadA/Sms might also contribute to synthesis-dependent strand annealing rather than canonical double-strand break repair.

    DisA physically interacts with RadA/Sms, that inhibits its diadenylate cyclase activity. This activity is also inhibited by HJ DNA or ssDNA, but the interaction of DisA with DNA and RadA/Sms is mutually exclusive. This could represent a mechanism to maintain c-di-AMP concentration during unperturbed growth but reduce its synthesis to signal the DNA damage as a checkpoint. DisA pausing at the site of DNA damage requires RecA and RecO activities. DisA physically interacts with RecA and modulates its ATPase and strand exchange activities to delay repair by recombination functions. Once DNA damage is repaired, the interaction of RecA with RadA/Sms and DisA may restore c-di-AMP synthesis and inactivate the checkpoint mechanism, to resume replication and cell proliferation.