Interactions Between the Cohesin Complex and Its DNA Partners: A Structural and Phylogenetic Approach
Open Access
- Author:
- Surcel, Alexandra
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 08, 2007
- Committee Members:
- Hong Ma, Committee Chair/Co-Chair
Wendy Hanna Rose, Committee Member
David Scott Gilmour, Committee Member
William O Hancock, Committee Member
Douglas Koshland, Committee Member - Keywords:
- Cohesin
CAR
Sister Chromatid Cohesion
SMC
Structural Maintenance of Chromosome
Yeast
Minichromosome Affinity Purification
MAP
Phylogeny
TEM - Abstract:
- Cohesin is an evolutionarily conserved protein complex responsible for maintaining sister chromatid cohesion from early S phase to the metaphase-anaphase transition. The cohesin complex is comprised of four proteins – Smc1 and Smc3 that heterodimerize, and Scc1 and Scc3. Imaging of the Smc heterodimer shows that it forms a V shaped molecule and imaging of the entire cohesin holocomplex suggests that it forms an enclosed ring structure. The cohesin complex binds to specific loci along the chromosome arms and centromeres known as Cohesin Attachment Regions (CAR). In lieu of consensus binding sequences at CAR loci, several models have been proposed for cohesin interactions at CAR sites, though no direct structural information about the in vivo interaction of cohesin at CARs has been obtained. This thesis is the first documented effort of the successful isolation and imaging of cohesin-chromatin complexes assembled in vivo. A minichromosome containing a CAR sequence and a CEN3 sequence was isolated from Saccharomyces cerevisiae using the Minichromosome Affinity Purification (MAP) method. The MAP protocol had to be overhauled to obtain high yield from this low-copy centromeric construct. Adjustments to MAP resulted in a 100-fold increase over the yield obtained by using the published protocol. Samples were isolated from G1 synchronized cultures in which cohesin is not bound to CARs and M-phase synchronized cultures in which cohesin is bound to CARs. These MAP isolated samples were negatively and positively stained for TEM analysis. Images showed that replicated minichromosomes always interact with one end of a flexible rod. Length measurements of this protrusion are consistent with a collapsed cohesin ring and width measurements of the rod suggest that multiple cohesins interact at CAR loci. These images lead to a model that suggests conformational changes within the cohesin complex may be responsible for the topological binding of cohesin to chromatin. Phylogenetic analyses were undertaken of the Structural Maintenance of Chromosome (SMC) family of proteins in an effort to identify conserved sequences within the arms of Smc1 and Smc3 that may explain the interaction observed via TEM along the coiled-coil domains of these molecules. Extra disruptions to the coiled-coil domains among SMC members were identified. These disruptions further support an alternative to the evolutionary history of these proteins that is presented in phylogenetic trees based solely on sequence alignments. In addition, phylogenetic analysis of the meiotic form of Smc1, known as Smc1b, suggests that it arose via a gene duplication event early in animal divergence, even though it has only been maintained in vertebrate lineages. This study of Smc1b lends support to the evolution of various meiotic regulatory mechanisms among animals.