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Título: | Biología estructural de la reparación de roturas de doble cadena en el ADN |
Autor: | Rivera-Calzada, Angel CSIC ORCID | Director: | Llorca, Óscar CSIC ORCID | Palabras clave: | DNA-PKcs Ku70 Ku80 NHEJ reparación del DNA microscopía electrónica biología estructural |
Fecha de publicación: | 2008 | Editor: | Universidad Complutense de Madrid | Resumen: | Double strand DNA breaks (DSBs) are one of the most challenging threats for cell
viability. The main mechanism to repair DSBs in higher eukaryotes is the process known as non-homologous end-joining (NHEJ) which consists in the ligation of the
generated ends after the break using no template. The components that participate in
this repair pathway play a fundamental role in maintaining genomic stability.
Furthermore they participate in programmed DNA breaks mediating biological
processes such as V(D)J recombination. During NHEJ the Ku protein binds to the
generated ends and recruits the catalytic subunit of the DNA Dependent Protein Kinase
(DNA-PKcs) to the DSB. The DNA-PKcs:Ku complex formed on a DNA end acts then
as a scaffold for the additional enzymatic activities required during the repair process.
Besides, the interaction of two DNA-PKcs:Ku:ADN complexes can create a synaptic
complex that holds both DNA ends together avoiding their diffusion. Some DSBs could
require the processing of the generated ends prior to the final ligation of both ends. The
nuclease Artemis has been implicated in this processing in association with DNA-PKcs
and, additionally, the DNA-PKcs:Artemis complex has been proposed to act as the
enzyme that opens the hairpin present in the coding ends during the V(D)J
recombination process.
The focus of my Ph.D. project consists in the structural determination of molecular
complexes implicated in the repair of DSB by NHEJ. To carry out this analysis I have
used structural techniques based on the combination of electron microscopy and single
particle analysis. More specifically I have solved the structure of full-length Ku alone
and after binding to DNA. These structures have provided a structural model that
explains the conformational changes that take place in Ku upon DNA end recognition
and their functional implications. Also, I have determined the structure of DNA-PKcs
that, combined with further analysis, has provided a structural model that explains the
overall topology of this kinase. The conformational changes that take place in DNAPKcs
upon recognition of DNA have also been characterised. Upon DNA binding
significant movements in key domains of DNA-PKcs seem to act as transducer of DNA
recognition to the catalytic domain. Additionally I have solved the structure of the DNAPKcs:
Ku:DNA complex providing important clues in the structural basis of the NHEJ
reaction. When analysing the DNA-PKcs:Ku:DNA complex the presence of a dimeric
complex constituted by two DNA-PKcs:Ku:DNA complexes facing each other was
observed. The structure of this dimeric complex is fully compatible with the requirements described for the synaptic complex that participates in the NHEJ process. Finally I have also determined the biochemical requirements to form a complex between DNA-PKcs and Artemis proteins in our experimental conditions. This analysis has provided the initial characterization required for future structural studies of the DNA-PKcs:Artemis complex. Overall, the results presented in this Thesis have contributed to our understanding of the structural basis of the NHEJ DNA repair reaction. The work provides the structural foundations of some of the biological functions and regulatory mechanisms of DNA-PKcs and Ku. |
Descripción: | Leída en la Universidad Complutense de Madrid. Facultad de Ciencias Biológicas el 07-09-2008; 318 págs. | URI: | http://hdl.handle.net/10261/42677 |
Aparece en las colecciones: | (CIB) Tesis |
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Rivera_Calzada_Angel.pdf | 48,03 MB | Adobe PDF | Visualizar/Abrir |
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