Isolation of centromeric DNA by utilizing the DNA binding activity of recombinant centromere proteins.

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Centromere/kinetochore is defined as the microtubule attachment site at the primary constriction of chromosomes. Electron microscopy studies have revealed that mammalian kinetochore has the trilaminar structure. However, the molecular basis of centromere/kinetochore structure has not been characterized extensively. It is assembled from the underlying centromere heterochromatin during the cell division cycle. Thus far, three major centromere autoantigens (CENtromere proteins ; CENP-A,CENP-B and CENP-C) together with HP1HSalpha, human homolog of Drosophila heterochromatin-associated protein 1, have been shown to be localized to centromere throughout the cell cycle. We have shown that they are structural components of centromere/kinetochre heterochromatin which possess DNA-binding activity in vitro and determined the DNA-binding domains. In this project, we further characterize the molecular interactions between DNA and CENPs further and identify novel centromere/kinetochore proteins that are associated with these CENPs in interphase and metaphase chromosomes by molecular biological and biochemical procedures.