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Functional characterization of nucleosome assembly proteins




Krzizike, Daniel, author
Luger, Karolin, advisor
Kennan, Alan, committee member
Nyborg, Jennifer, committee member
Stargell, Laurie, committee member
Woody, Robert, committee member

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The amount of DNA found within the human body will span from the earth to the sun ~50 times. With the DNA providing the genetic blueprint of all living things, it needs to be packaged in a way that allows accessibility. The first step in this packaging involves nucleosomes, large macromolecular complexes made up of histone proteins and DNA. Nucleosomes must remain dynamic as they are constantly assembled and disassembled for processes such as DNA replication, repair, and transcription. Both assembly and disassembly occur in a specific stepwise manner orchestrated by multiple proteins employed by the cell. Specifically, histone chaperones have been implicated in almost every aspect of nucleosome dynamics such as shuttling histones into the nucleus, histone storage, and both nucleosome assembly and disassembly in an ATP-independent manner. While the structures of many histone chaperones have been determined, the mechanism of how they regulate nucleosome dynamics is still largely unknown. I investigated the mechanism of the nucleosome assembly protein family (Nap family) through several biochemical approaches. The Nap family of proteins are implicated in histone homeostasis through interactions with core histones, histone variants, and linker histones. They are conserved among all eukaryotes from yeast to humans. Members of the Nap family contain a conserved core region flanked by highly disordered N- and C-terminal tails varying in length and charge between species. Using yNap1, we investigated how these tails impact the overall function in regards to histone binding, histone selectivity among core histones and histone variants, and in mediating histone-DNA interactions. We found that the tails are critical for overall function, with the charge of the tails being crucial in regulation. We also investigated Vps75, another member of the Nap family. Similar to Nap1, Vps75 binds core histones, but also stimulates the acetylation activity of Rtt109, a histone acetyltransferase. In light of a recent debate regarding the stoichiometry with which these Nap members bind their histone cargo, we characterized the Vps75-histone interaction using core histones H2A-H2B and H3-H4. Comparing Vps75 with yeast Nap1, we found that the mechanism of histone binding is not conserved among these Nap family members. Further expanding on Vps75, we investigated the interaction with Rtt109 in both the presence and absence of H3-H4. We discovered dimeric Vps75 is capable of binding either one histone tetramer or two units of Rtt109 with the ternary complex consisting of only one unit of Rtt109 and one H3-H4 tetramer. While characterizing Nap family members I became very familiar with Analytical Ultracentrifugation (AUC). AUC is a powerful in-solution technique that provides first-principle hydrodynamic information to determine size, shape, and molecular interactions, making it ideal for the characterization of proteins, DNA, and the interactions among them. As our lab traditionally used AUC to obtain van Holde-Weischet plots, an excellent graphical representation of homogeneity or heterogeneity, we incorporated new analysis techniques for improved accuracy in molecular mass and gross shape determination. Using the added-on fluorescence detection system, we obtained a level of sensitivity and selectivity that was otherwise not possible. Using the powerful method of analytical ultracentrifugation combined with fluorescent studies, we provide insight into the regulation mechanism of Nap family members along with establishing a framework to study other macromolecular complexes.


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