The human kinetochore: model building and data from fluorescence microscopy

S. Diekmann, J. Sühnel, S. Orthaus, S. Weitkamp-Peters, P. Hemmerich
IMB, Beutenbergstr. 11, 07745 Jena

At each cell division (mitosis), accurate segregation of every DNA chromosome into the two daughter cells is ensured by the assembly of a kinetochore multi-protein complex at each centromere locus. In humans, the centromere is a multifold repeat of α-satellite DNA, each 171 base-pairs in length forming one nucleosome. Centromere nucleosomes are distinct from normal nucleosomes by the exchange of the histone H3 to CENP-A which is very similar in sequence and length. On this repeated structure, several "founding" kinetochore proteins settle. If the α-satellite DNA is deleted from the chromosome, the kinetochore multi-protein complex settles on another stretch of DNA of the chromosome. Thus, there seems to be no direct DNA signal for the complex to form but instead - may be - structural properties.
Our geometrical model building studies of multi-nucleosomal chromatin built of nucleosomes and linker DNA (the DNA between the nucleosomes) indicate that structures without sterical clashes can be formed only for specific linker lengths. Some data suggest that the most compact of these structures is the α-satellite DNA with 171 bp repeat length. By model building, we now place the founding kinetochore proteins onto this structure. By measuring the protein interactions in vitro as well as in the human cell, we experimentally confirm specific details of the model. We measure local distances between GFP-labelled kinetochore proteins by fluorescence microscopy (FRET and FLIM).
From these studies we hope to learn why the kinetochore founding complex settles on α-satellite DNA.