Tesis doctoral de Marta Rajkiewicz
A crucial step in dna replication is opening of the ring-shaped sliding clamp, and its proper placement on the dna template. This process is carried out by the clamp loader complexes, in an atp dependent reaction that is stimulated by the primed dna. In the gram-negative bacteria e. Coli, two main components of the clamp loader are ¿ and ¿, products of the same dnax gene, that isolated in solution, form homotetramers. In the presence of additional subunits ¿ and ¿¿, the equilibrium shifts and the (dnax)3¿¿¿ is formed. Other two subunits, ¿ and ¿ , form a complex with the clamp loader. The ¿ subunit binds to the dnax proteins (¿ or ¿) on one side and to ¿ on the other. The ¿ subunit forms a bridge between (dnax)3¿¿¿and ¿ links the clam loader to ssb facilitating the displacement of primase from newly synthesized rna primer, thus playing a crucial role in okazaki fragment synthesis. we have analyzed the structure of e. Coli ¿4 and ¿4¿¿ complexes using three-dimensional electron microscopy. Reaching convergence in 3d map refinement was hindered by several reasons: the small size (165 kda to 222 kda), lack of symmetry, and globular shape of the complexes, which resulted in low signal in the images; and large structural heterogeneity, which could be caused by intrinsic complex flexibility, and was refractory even to sample cross-linking procedures. Nevertheless, by using maximum-likelihood image classification methods we were able to select homogenous subpopulations of particles and calculate consensus three-dimensional maps for all complexes. Combination of our maps with crystallographic or homology model structures show how dnax proteins can establish a large variety of interactions among them, in agreement with their versatility in forming complexes with different accessory proteins during polymerase holoenzyme assembly. Difference maps show the location of the ¿¿ heterodimer in the ¿4¿¿ complex, and how the interactions among ¿ subunits are altered by its presence.
Datos académicos de la tesis doctoral «Structures studies on e.coli dna polymerase iii clamp loader subcomplexes.«
- Título de la tesis: Structures studies on e.coli dna polymerase iii clamp loader subcomplexes.
- Autor: Marta Rajkiewicz
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 22/10/2010
Dirección y tribunal
- Director de la tesis
- José María Carazo Garcia
- Tribunal
- Presidente del tribunal: jose Lopez carrascosa
- oscar Antonio Llorca blanco (vocal)
- Rafael Núñez ramírez (vocal)
- mikel Valle rodriguez (vocal)