USE OF YTH TO IDENTIFY INTERACTIONS WITH CENTROMERIC NUCLEOSOMES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The eukaryotic genome is packaged into nucleosomes, particles that consist of DNA wrapped around histone proteins. Centromeres are specialized regions of chromatin that serve as points of attachment of kinetochore during mitosis. In the course of recent work it has been proposed that the structure of the centromeric histone core is fundamentally different than that of the canonical one. Canonical nucleosomes contain two molecules each of histones H2A, H2B, H3 and H4 that together comprise an octamer. However, recently obtained experimental evidence suggests that centromeric nucleosome is a smaller particle, most likely containing a single copy of histones H2A, H2B, CenH3 and H4, a hemisome. The hemisome model predicts that novel surfaces on the CenH3-containing particle that are not exposed on the canonical particle can be specifically bound by kinetochore proteins. Using the crystal structure of the canonical nucleosome as a starting point we have identified regions of H2B and CenH3 that might be exposed to solvent in a hemisome and thus be available for binding by kinetochore proteins. We would like to use the Yeast Two Hybrid system to screen for interaction partners of these proteins, using histone H4 as a control. We hope to identify proteins with known kinetochore function that will test our model and allow us to propose new hypotheses for the inner kinetochore structure.