STRUCTURAL INVESTIGATION OF CENP-A CONTAINING NUCLEOSOMES UTILIZING SINGLE-MOLECULE FÖRSTER RESONANCE ENERGY TRANSFER
Open Access
Author:
Sennett, Michael A
Graduate Program:
Chemistry
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
October 28, 2014
Committee Members:
Tae Hee Lee, Thesis Advisor/Co-Advisor
Keywords:
smFRET nucleosome CenP-A
Abstract:
Interest in how a functional kinetochore assembles at the centromere has grown in recent years as a result of our increased understanding of the chromosome. Centromere Protein A (CenP-A) is a histone H3 variant and is believed to play an essential role in defining the location of the centromere, rather than the DNA sequence. Based on recent crystal structures and models of nucleosomes containing Cenp-A, it has been hypothesized that the structures of Cenp-A nucleosomes is different from that of canonical nucleosomes. , Using single-molecule Förster resonance energy transfer (smFRET), I tested the hypothesis in vitro using physiologically relevant salt conditions. smFRET measurements reveal that the structure of fluorescently labeled DNA in complex with CenP-A containing centromere nucleosomes is different from the canonical nucleosomes. This indicates a specific role for CenP-A variants, suggesting a basis for the mechanism of centromere identification and kinetochore assembly.
