RECONSTITUTION OF THE YEAST CENTROMERIC NUCLEOSOME CORE PARTICLE

Open Access
Author:
Friedland, Daniel Bakke
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan , Thesis Supervisor
  • Stephen Schaeffer, Honors Advisor
Keywords:
  • molecular biology
  • yeast
  • Cse4
  • centromere
  • chromatin
  • nucleosome
  • HPLC
  • reconstitution
Abstract:
The centromere constitutes the locus on the chromosome onto which the kinetochore assembles and microtubules attach to direct separation of sister chromatids during mitosis. Proper formation of the centromere-kinetochore complex is essential for accurate cell division as abnormalities in the cell cycle are associated with many diseases, including cancer. Thus, understanding the structures and mechanisms of the centromere-kinetochore complex may provide insight into disease pathogenesis. Centromeric DNA, along with all nuclear DNA, is stored in a highly condensed, organized structure known as chromatin. Chromatin consists of a series of octameric protein complexes around which the DNA is wrapped. The complex of this octamer and its associated DNA is called the nucleosome core particle. Canonical nucleosome core particles are comprised of two copies each of H2A, H2B, H3, and H4, but centromeric nucleosomes contain a variant, CenH3 in place of H3. Nucleosomes serve as the substrate for several post-translational modifications, which change chromatin structure, facilitate interactions with chromatin enzymes, and regulate access to certain genes for transcription. The nucleosome serves as a fundamental model for studying gene regulation. Unlike the centromeres of most eukaryotes, the budding yeast centromere contains only one nucleosome. This makes the yeast centromeric nucleosome an ideal model for centromere studies. In this project, a yeast centromeric nucleosome was created for potential use in binding studies with the kinetochore proteins. Additionally, parameters for further reconstitutions of this nucleosome were determined.