Analysis of chromosome condensation in Saccharomyces cerevisiae.
Doctoral thesis, UCL (University College London).
Eukaryotic chromosomes reach their stable rod-shaped appearance in mitosis in a reaction dependent on the evolutionarily conserved condensin complex. It is currently unknown how and where condensin associates with chromosomes. Here, we analyse condensin binding to budding yeast chromosomes by immunoprecipitation followed by hybridization on high resolution oligonucleotide tiling arrays. We observe that condensin binding sites coincide with those of the RNA polymerase III transcription factor TFIIIC and the loading factor Scc2/4 of the related cohesin complex. Both TFIIIC and Scc/4 facilitate condensin loading onto chromosomes. An isolated ectopic B box motif is sufficient to prime formation of a condensin binding site. While cohesin translocates away from these loading sites, condensin persists there. This defines the loading sites of cohesin and condensin and explains how an alternating pattern of these complexes along chromosomes is established. The findings have important implications for the fields of chromosome segregegation and nuclear structure in interphase and mitosis. The identification of SMC loading sites will allow targeted probing of eukaryotic chromosomes. Beside condensation, mitotic chromosome segregation also depends on the chromosomal condensin complex. Without condensin, sister chromatids fail to resolve causing anaphase bridges and chromosome breakage. How condensin promotes sister chromatid resolution is unknown. We have used the budding yeast rDNA as a model locus, whose segregation depends on condensin activity during anaphase. We show that anaphase bridges in a condensin mutant are resolved by ectopic expression of a foreign (Chlorella virus) but not endogenous yeast topoisomerase II (topo II). This suggests that catenation prevents sister rDNA segregation, and that yeast topo II is ineffective in decatenating the rDNA in the absence of condensin. We furthermore find that expression of Chlorella virus topo II in wild type cells advances the normally late segregation timing of the rDNA locus. This suggests catenation is a mean for the cell to provide rDNA cohesion up to late anaphase, when condensin promotes decatenation. This provides the first direct evidence of condensin's role in the disengagement of topologically connected sister chromatids.
|Title:||Analysis of chromosome condensation in Saccharomyces cerevisiae|
|Additional information:||Pending digitisation|
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