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DNAMEREP Report Summary

Project ID: 614541
Funded under: FP7-IDEAS-ERC
Country: Italy

Mid-Term Report Summary - DNAMEREP (The role of essential DNA metabolism genes in vertebrate chromosome replication)

Genome duplication is a key event in the life cycle of all biological organisms and its careful control is essential to preserve the physical integrity of chromosomes. Maintenance of genome integrity during DNA replication relies on several factors such as the ability to respond to DNA lesions impairing fork progression, the coordination of DNA repair and DNA replication and the availability of DNA replication origins. These mechanisms are particularly important for the replication of specific chromosome regions with complex or repetitive DNA sequence composition, which are often involved in mutational events responsible for several human diseases. To uncover the role of essential DNA metabolism genes in chromosome replication in higher eukaryotes we set up several novel assays to: 1) Characterize molecular mechanisms involved in the assembly and distribution on chromatin of replication origins in vertebrate cells; 2) Uncover the role of essential DNA recombination and fork protection proteins in chromosomal DNA replication; 3) Identify and characterize factors required for faithful replication of complex vertebrate genomic regions
The mechanisms underlying the function of essential DNA repair proteins in unchallenged chromosomal DNA replication are poorly understood. This is in part due to the fact that many of the genes involved in DNA metabolism are essential for cell viability, especially in higher eukaryotes. As these functions are essential for cell duplication and survival the study of their integrated functions at molecular level benefits from the use of biochemical model systems and single molecule analysis tools. Cell free systems based on vertebrate Xenopus laevis egg extract have been helpful to elucidate the biochemical bases of cell cycle checkpoints, DNA repair and DNA replication. These cell free-systems can reproduce basic cell cycle events such as chromatin formation, nuclear assembly, semi-conservative DNA replication and mitosis. The high degree of genetic conservation between Xenopus and mammalian organisms facilitates the study of large proteins present in egg extracts. Specific protein depletion from egg extract can facilitate the characterization of the biochemical steps in which essential DNA metabolism proteins operate, allowing the study of essential vertebrate genes that would normally compromise viability when inactivated in other cellular systems, especially during chromatin replication. Using this system we reconstituted for the first time the replication, the protein composition and the structure of the chromatin associated highly repetitive DNA contained in human centromeres. Our approach revealed striking new features of centromeric chromatin. To achieve this goal we developed innovative methods based on Electron Microscopy (EM) to analyze DNA molecules undergoing replication and repair reactions. Using EM on centromeric chromatin we showed for the first time the tri-dimensional structure of the centromeric chromatin, which forms large double stranded DNA loops embedded in a protein matrix made of condensin proteins. Using a similar approach to visualize replication intermediates we have characterized the role of RAD51 and BRCA2 in genomic DNA replicated in extracts depleted of these proteins.

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