Date of Graduation

8-2016

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Pinto, Ines

Committee Member

McNabb, David S.

Second Committee Member

Thallapuranam, Suresh

Third Committee Member

Evans, Timothy A.

Keywords

Biological sciences; Chromosome segregation; Histones; Yeast

Abstract

Nucleosomes, the basic unit and the building blocks of chromatin have an essential role in the tight packaging of DNA into higher order chromatin. Work from our lab and others have provided information on the contributions of different histone proteins and specific domains within the nucleosome made to create the centromeric chromatin structure required for normal chromosome segregation during mitosis. The DNA entry/exit site is a particular region of the nucleosome where histone H2A, H3 and H4 form critical interactions that appear to be essential for the association of Sgo1, a tension sensing protein that monitors kinetochore-microtubule attachment. In our study, we first characterized histone H2A mutants with respect to their chromosome segregation phenotypes. Three mutations that show such phenotypes were in the C-terminal region of H2A, which is located in the DNA entry/exit region of the nucleosome, in close proximity to H3 and H4 residues that show severe chromosome segregation defects when mutated. We then created a double mutant strain that incorporated two single mutations, one in H2A and one in H4, to study their combined effect in chromosome segregation and normal cell cycle progression. The H2A N115S residue, positioned in the C-terminal tail of histone H2A, and H4 K44Q, positioned in H4 L1 histone fold domain, both falling in the region of DNA entry/exit point of the nucleosome, were incorporated in our double mutant. We found that the incorporation of the H2A N115S mutation alleviated the growth defect of the H4 K44Q single mutant and fully suppressed its increase-in-ploidy phenotype. We also found that overexpression of Sgo1 suppressed the sensitivity of both single mutants and the double mutant to the microtubule depolymerizing drug benomyl. We conclude that histone-histone interactions within the DNA entry/exit point of the nucleosome are particularly important in chromosome segregation, most likely in establishing centromere-kinetochore attachments during mitosis. In addition, and consistent with a previously noted role of the DNA entry/exit point, this nucleosomal region creates a unique surface required for the recruitment of Sgo1, and perhaps other proteins, such as components of the CPC, required for normal microtubule attachment at the centromere.

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