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A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast

Nature Cell Biology volume 11, pages 1109–1115 (2009)Cite this article

Abstract

Kinetochores are large multiprotein complexes that mediate chromosome segregation in all eukaryotes by dynamically connecting specialized chromosome regions, termed centromeres, to the plus-ends of spindle microtubules1,2. Even the relatively simple kinetochores of the budding yeast Saccharomyces cerevisiae consist of more than 80 proteins, making analysis of their respective roles a daunting task3. Here, we have developed a system that allows us to artificially recruit proteins to DNA sequences and determine whether they can provide any aspect of kinetochore function in vivo. We show that artificial recruitment of the microtubule-binding Dam1 complex to a plasmid lacking any centromere DNA is sufficient to confer mitotic stabilization. The Dam1-based artificial kinetochores are able to attach, bi-orient and segregate mini-chromosomes on the mitotic spindle, and they bypass the requirement for essential DNA-binding components of natural kinetochores. Thus, we have built a simplified chromosome segregation system by directly recruiting a microtubule force-transducing component to DNA.

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Figure 1: A strategy to construct artificial kinetochores in S. cerevisiae.
Figure 2: Live-cell imaging of mini-chromosome segregation mediated by Ask1–and Dam1–TetR.
Figure 3: Analysis of artificial kinetochore function.
Figure 4: The artificial kinetochore can direct the segregation of a native yeast chromosome.

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Acknowledgements

The authors wish to thank all members of the Westermann lab for discussions, and Jan-Michael Peters and Barry Dickson for critical reading of the manuscript. We thank Soni Lacefield and Andrew Murray for communicating results before publication, and the Nasmyth and Drubin/Barnes labs for strains and plasmids. Research in the laboratory of S.W. was supported by the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC starting grant agreement no. [203499], and by the Austrian Science Fund FWF (SFB F34-B03).

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  1. Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, Vienna, 1030, Austria

    Eva Kiermaier, Sophie Woehrer, Karl Mechtler & Stefan Westermann

  2. Department of Molecular and Cell Biology, University of California, Berkeley, 16 Barker Hall, Berkeley, 94720-3202, USA

    Yutian Peng

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Contributions

S.W. and E.K. designed the experiments and wrote the manuscript. E.K. performed most of the experiments with help from So. W., Y.P. and S.W; K.M. contributed the mass spectrometry analysis.

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Correspondence to Stefan Westermann.

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Kiermaier, E., Woehrer, S., Peng, Y. et al. A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast. Nat Cell Biol 11, 1109–1115 (2009). https://doi.org/10.1038/ncb1924

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