Abstract
In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin.
Highlights
Centromeres were classically defined as the primary constriction of chromosomes—a region where paired sister chromatids are held tightly together until the onset of anaphase
It is generally accepted that the underlying DNA sequence that defines the primary constriction of mitotic chromosomes is the centromere, whereas the proteinaceous structure that assembles on the surface of the centromeric chromatin is the kinetochore
The distribution of kinetochore proteins along the DNA has been mapped at vertebrate neocentromeres by a number of methods (Alonso et al 2007; Shang et al 2013; Saffery et al 2003; Capozzi et al 2008), but it is not known how much DNA is folded into a functional mitotic kinetochore
Summary
Centromeres were classically defined as the primary constriction of chromosomes—a region where paired sister chromatids are held tightly together until the onset of anaphase. Classic microscopic observations revealed that this was the region where mitotic chromosomes attached to the spindle microtubules. There was an ongoing debate about the distinction (if any) between centromeres and kinetochores. Both terms were initially proposed to describe essentially the same aspect of chromosome structure and function, so this seemed liked a semantic discussion. It is generally accepted that the underlying DNA sequence that defines the primary constriction of mitotic chromosomes is the centromere, whereas the proteinaceous structure that assembles on the surface of the centromeric chromatin is the kinetochore. The histone H3 variant CENP-A is a specific protein marker for centromeres (Earnshaw and Rothfield 1985; Earnshaw et al 2013), whereas kinetochores are characterized by the presence of >100 different proteins, the earliest described being CENP-C (Earnshaw and Rothfield 1985; Saitoh et al 1992)
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