All kinetochores are megadalton protein structures that assembled on centromeric chromatin to form a single microtubule-binding site. Kinetochores are assembled from conserved protein subcomplexes. Although a number of models of kinetochore structure have been proposed based on protein-protein interaction experiments and physical studies of individual subcomplexes, little is known about the architecture of an intact kinetochore and the mechanism by which kinetochores bind to and maintain attachments to dynamic microtubules. In addition, the post-translational modifications required for kinetochore assembly are not known. To address these issues, we have developed a method to assemble kinetochores de novo and are using this to dissect the assembly process. We are also pursuing kinetochore structure using EM techniques.
We are performing electron microscopy to elucidate the overall architecture of kinetochores purified from yeast.
We have developed an assay for kinetochore assembly in vitro using a centromeric DNA template. We are using this assay to identify the post-translational modifications required for assembly and using a single molecule assay to monitor kinetochore assembly in real time.