Cryo-EM study of inner kinetochore explains how it recognises centromeric DNA and functions as a load-bearing element during mitotic chromosome segregation
Structure of the inner kinetochore bound to centromeric CENP-A nucleosome
Organism survival is dependent on the propagation and passage of its genetic information. DNA is condensed and packaged into chromosomes during mitosis and specific regions on chromosomes, known as centromeres, are responsible for segregating sister chromatids to two daughter cells. In humans, centromeres – the constricted region of DNA on a chromosome – are composed of thousands of copies of a 171 base pair (bp) α-satellite DNA repeat sequence. Centromeres recruit kinetochores, large multi-protein complexes, that are responsible for the attachment of chromosomes to the mitotic spindle. Kinetochores assemble onto specific centromere protein A (CENP-A) nucleosomes that are installed onto 171 bp α-satellite repeats of centromeric DNA. The inner kinetochore is attached to centromeres to hold onto DNA, whilst the outer kinetochore attaches to microtubules which pull the two strands of replicated DNA apart into two daughter cells during mitosis. How human kinetochores recognise centromeric CENP-A chromatin, and how they establish a robust connection between the chromosome and the mitotic spindle has so far remained a mystery. A new study from David Barford’s group in the LMB’s Structural Studies Division, has solved the structure of the human inner kinetochore when bound to a CENP-A nucleosome, and shed new light on how the kinetochore interacts with centromeric chromatin.
Lead authors Stanislau Yatskevich and Kyle Muir prepared components of recombinant kinetochores and assembled them onto CENP-A nucleosomes, in order to map them using cryogenic electron microscopy (cryo-EM). Their principal finding was the mechanism by which the inner kinetochore constitutive centromeric associated network (CCAN) complex assembles onto the centromeric CENP-A nucleosomes of the 171 bp α-satellite repeats. Contrary to prevailing models, the group discovered that the 16 subunits of CCAN form a defined globular complex, which includes a central tunnel that perfectly complements the shape and charge of duplex DNA.
The newly determined structure showed that, when bound to CENP-A nucleosomes on α-satellite repeats, the linker DNA between adjacent nucleosomes is topologically entrapped by CCAN. Much like a hand clasping a rope, CCAN grips approximately 46 bp of DNA through its central tunnel. A further 125 bp of DNA wrap the CENP-A nucleosome, thereby explaining the conservation of the 171 bp repeat. The tight grip on DNA by CCAN creates a robust load-bearing device, which ensures it remains attached to chromosomes and withstands the mitotic spindle’s pushing and pulling forces throughout chromosomal segregation.
Further examination also showed that some of the subunits comprising CCAN are histone-fold proteins. These proteins bind linker DNA in a manner similar to canonical histones of nucleosomes.
This research improves our understanding of mitosis and the manner in which DNA inheritance is preserved. These new insights into how the kinetochore functions could prove important to later clinical research, as errors in chromosome segregation (as mediated by kinetochores) can cause aneuploidy – the basis of numerous genetic diseases, including developmental disorders and cancers.
The work was funded by UKRI MRC, Cancer Research UK and Boehringer Ingelheim Fonds.
Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome. Yatskevich, S., Muir, KM., Bellini, D., Zhang, Z., Yang, J., Tischer, T., Predin, M., Dendooven, T., McLaughlin, SH., Barford, D. Science
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