Papel de las proteínas RuvA, RuvB y RecU en las etapas tempranas y tardías de la recombinación homóloga en Bacillus subtilis

• Autores: Cristina Cañas Muñiz
• Directores de la Tesis: Silvia Ayora Hycsh (dir. tes.), Juan Carlos Alonso Navarro (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2011
• Idioma: español
• Tribunal Calificador de la Tesis: José Berenguer Carlos (presid.), Luis Blanco Dávila (secret.), Óscar Fernández Capetillo (voc.), Enrique Viguera Mínguez (voc.), José Rafael Penades Casanova (voc.), J. Pablo Radicella (voc.), Carmen San Martín Pastrana (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: digitool-uam.greendata.es
• Resumen

  • DNA homologous recombination is an essential process required for the generation of biological diversity, the maintenance of genomic stability after DNA-damage, and the restart of stalled replication forks. After end processing and as a result of the strand invasion activity of the RecA recombinase, a recombination intermediate (termed Holliday junction, HJ) is generated. This intermediate is formed by two recombining DNA molecules linked by a crossover. In B. subtilis the complex that processes the HJ in order to obtain two independent DNA molecules (monomeric chromosomes) is composed by the RuvAB translocase and the RecU HJ resolvase. Mutations in the ruvA, ruvB or recU genes (epistatic group ¿) render cells very sensitive to DNA damaging agents, as methylmethane sulfonate (MMS).

    The active RecU resolving enzyme, which is a dimer in solution, consists of two monomers of 206 residues. The protein contains a large and flexible N-terminus (residues 1-33), which is responsible of the stability of the HJ-RecU complex and the interaction with RuvB and contains the highly conserved NRGM tetrad. The residue R31 of the NRGM tetrad is directly implicated in the interaction between both proteins. The N-terminus is essential for the catalytic activity of RecU and for proper chromosome segregation. The expression of recU, which is in the recU-ponA operon, is regulated by the ¿M regulon. The expression of recU is induced under different membrane stresses, included addition of vancomycin. In contrast, the recU expression is not induced during the SOS response.

    RecU is able to modulate RecA activities, which suggests a role in early steps of the HR process. In this work, two mutants of RecU, recU56 and recU71, have been characterized. These mutants promote resolution of HJs, but do not promote RecA modulation. This RecU activity plays an important role in vivo, since both mutants are very sensitive to MMS and are impaired in plasmid transformation. During plasmid transformation, which is a RecA-independent process, RecU would modulate the formation of RecA presynaptic filaments that might be deleterious in a ¿recU context. By this way, the absence of RecA, suppresses the RecU requirement during plasmid transformation. A RecU-HJ model was proposed. In this model the stalk region, which is highly conserved among RecU proteins, would be accommodated in the central hole formed in the HJ. Two aromatic residues, Y80 and F81, are directly implicated in the specific recognition of HJ DNA and HJ distortion. RecUY80A and RecUF81A fail to interact and modulate RecA activities, which supports the idea that the stalk region is implicated in RecA interaction. Both activities, RecA modulation and HJ binding, are mutually exclusive, and once RecU interacts with a HJ, does not interact with RecA.

    RuvA and RuvB catalyze the HJ branch migration. RuvA, which forms tetramers u octamers and binds at the centre of the HJ, stimulates the ATPase activity of RuvB and it is essential for helicase activity. RecU and RuvB interact in vitro, and form two types of complexes at the center of the HJ, detected by AFM. RuvB stimulates the RecU HJ cleavage favouring a symmetrical resolution, which is essential for the proper segregation of the two DNA molecules. RuvA is able to displace RecU from the RecU-RuvB-HJ complex and to inhibit RecU HJ cleavage. However the branch migration activity of RuvAB is needed to reach the RecU recognition sequence at the junction point.