Date of Award

2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Medicinal Chemistry and Molecular Pharmacology

First Advisor

Tony R. Hazbun

Committee Chair

Tony R. Hazbun

Committee Member 1

Chang-Deng Hu

Committee Member 2

Douglas Lacount

Committee Member 3

Ruben C. Aguilar

Abstract

Precise and faithful segregation of chromosome segregation during mitosis depends on the ability of the cell to regulate chromosome bi-orientation on the mitotic spindle. Shugoshin (Sgo1), the protector of meiotic centromeric cohesin, is required for proper establishment of chromosome bi-orientation. Sgo1 plays a crucial role as part of a mitotic tension sensor between sister chromatids. Recently, Sgo1 has been reported to interact with histone H3 at the pericentromere region, as an important factor for tension sensing and chromosome segregation. However, the role of Sgo1 in tension sensing at centromere is still elusive. The centromere is the region of attachment of chromatin fiber to mitotic spindle via the kinetochore and these structures assist in segregation of chromosomes to opposite spindle poles during mitosis. Cse4, budding yeast centromere specific histone variant, is thought to substitute histone H3 when assembling into a centromeric nucleosome. Cse4 plays key roles in kinetochore formation and proper chromosome segregation. Cse4 contains conserved C-terminal histone fold domain and unique 135-amino-acid N-terminal tail that extends from the nucleosome core making it accessible to interacting proteins and modification. To date, there is no evidence of a direct physical interaction reported between the Cse4 tail and the kinetochore or cell cycle related-proteins.

In our study, we first established a direct interaction between the Cse4 N-terminal tail and Sgo1 by using an in vitro pull down assay. Sgo1 has a strong ability to associate with Cse4 tail, while it is not able to bind with another kinetochore protein tail, Cnn1, indicating the specificity of Sgo1-Cse4 interaction. From our kinetic binding study, interaction between Sgo1 and N-terminal tail of Cse4 has an equilibrium dissociation constant (KD) of approximately 33 nM. Moreover, we identified the minimal region on Cse4 tail (residue 49-65) that is sufficient for associating with Sgo1. Interestingly, part of this binding motif (residue 49-56) is conserved from present throughout eukaryotes. Furthermore, our pull down analysis and multiple sequence alignment analysis of Cse4 tail homologues suggest that there is an additional conserved motif, located within residues 95-102 of Cse4-tail, that is responsible for the Sgo1 interaction. In addition, an N-terminal proteolytic fragment of Sgo1 can interact with Cse4. The finding of the Sgo1 binding motifs, present in Cse4, suggests an attractive model in which the orthologous interaction is conserved in higher eukaryotes and this interaction could have an important role in tension sensing throughout the eukaryotic kingdom .

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