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.

Leída en la Universidad Complutense de Madrid. Facultad de Ciencias Biológicas el 07-09-2008; 318 págs.

Peer reviewed