Chromosomes are organized into morphologically distinct higher-order structures during chromosome segregation, DNA repair, and transcription. These different structures are mediated by structural maintenance of chromosome (smc) complexes that consist of 4 protein subunits conserved from bacteria to man. Smc complexes are thought to tether together two distinct regions of chromatin, either within the same DNA strand or between two distinct strands. However the mechanism for how they bind chromatin or tether it, are still very poorly understood. One Smc complex, cohesin, holds together the two sister chromatids (the newly replicated chromosomes) from the time of their synthesis in S phase through metaphase of mitosis. This sister chromatid cohesion is critical to ensure that daughter cells inherit proper ploidy during cell division. Cohesin is also important for mitotic condensation, meiotic chromosome structure, post-replicative DNA repair and the regulation of gene expression. Defects in cohesin and cohesin auxiliary factors are the cause of several human disorders and potentially contribute to chromosome instability associated with tumorigenesis. In this proposal I outline an integrated approach using genetics, cell biology, and new in vitro biochemical assays to study the establishment of cohesion using budding yeast. We will use a novel system to generate strains with serial quantized reduction in total cellular cohesin. Analysis of these strains will provide insights into the level and localization of cohesin that is needed in vivo to mediate its known diverse roles in chromosome metabolism as well as uncover potentially novel biological functions. We will investigate the role of ATP, cohesin loader and a cohesin antagonist in cohesin binding to chromosomes by exploiting dominant negative phenotypes of cohesin's Smc3 ATPase and by suppression analysis of its defective loader. We will elucidate the mechanism of converting chromatin bound cohesin to its cohesive state. Experiments in vivo will demonstrate the contribution of cohesin subunits to cohesion generation distinct from chromatin binding. We will also test whether cohesiveness reflects stabilization of the cohesin ring around the sister chromatids or oligomerization between cohesin complexes bound to each sister chromatid. The mechanism of cohesin binding to chromosomes will also be analyzed by a physiological-relevant in vitro assay for cohesin binding to chromatin that we have developed recently. Using this assay we will assess the contribution of DNA sequence, DNA topology, ATP, cohesin itself, and cohesin auxiliary factors to the cohesin-DNA complex formation. We will develop a relevant in vitro assay for cohesion using as a foundation our in vitro assembled cohesin-DNA complexes and our battery of cohesin mutations. Finally, the ability to assemble in vitro cohesin/DNA complexes and cohesive cohesin/DNA complexes will allow us to determine their ultrastructure in the electron microscope.
DNA, the hereditary material of chromosomes, is coated first by core histone proteins and then packaged into higher order structures by specialized protein complexes, called SMC complexes, much the way the magnet tape in a cassette is packaged by its plastic housing. Chromosome structures mediated by Smc complexes ensure that each newly form cell in an organism inherits the complete complement of chromosomes and that damage to the DNA of the chromosomes can be repaired efficiently. Mutations in human genes encoding Smc proteins have been linked to cancer, birth defects and disorders.
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