Organization of ’s clusters

Pierre Therizols, Bernard Dujon and Emmanuelle Fabre Unite de Genetique Moleculaire des Levures (URA 2171 Centre National de la Recherche Scientifique, UFR 927 Universite Pierre et Marie Curie), Departement Génétique et Genomes, Institut Pasteur, 75724 Paris Cedex, France

In the Saccharomyces cervisiae, the 32 cluster at the nuclear periphery in 6 to 8 groups, forming discrete focal compartments in which silencing factors (Sir2, Sir3, and Sir4) accumulate. We have shown in a previous work that the organization of subtelomeric regions was dependent on the integrity of a nucleopore’s subcomplex, i.e. Nup84 (Thérizols et al. J Cell Biol.;172(2):189-99). The tethering of telomeres, the subtelomeric silencing and the efficiency of repair of a DNA double-strand break generated in the subtelomeric region, are reduced in the absence of one or the other of the complex. We are currently investigating the composition of subtelomeric foci, identifying the involved in their formation, and if the Nup84 complex has a role on this. As a first step we have inserted, in wild type strains and different mutants of the Nup84 complex, the 5’ end of a truncated lys2 gene in the of chromosome XI-L and the 3’end in an other subtelomere ie 31 strains for all the possible combinations. We will measure the frequency of apparition of [lys+] colonies. The efficiency of the recombination could reflect the clusters organisation. To verify this hypothesis, we have as well inserted two hundred copies of the tetO operator at close proximity of the 3’lys2 cassette and one hundred copies of lacO operator close to the 5’lys2 cassette. With the insertion of tetR-GFP and lacI-GFP in the genome, we should be able to realize pair wise localization of each telomere in the nucleus in order to confirm the previous observation.