Recruiting a microtubule-binding complex to DNA directs chromosome segregation in budding yeast. Nat Cell Biol

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Nature Cell Biology (Impact Factor: 19.68). 09/2009; 11(9):1116-20. DOI: 10.1038/ncb1925
Source: PubMed


Accurate chromosome segregation depends on the kinetochore, which is the complex of proteins that link microtubules to centromeric DNA. The kinetochore of the budding yeast Saccharomyces cerevisiae consists of more than 80 proteins assembled on a 125-bp region of DNA. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1-DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, bi-orient sister kinetochores at metaphase during the mitotic cycle, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore functions do not depend on the DNA-binding components of the natural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers assembly of the complex structure that directs mitotic chromosome segregation.

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    • "Strains Strains are listed in Table S1. Synthetic kinetochore strains were previously described (Lacefield et al., 2009). The P CSE4 GFP-CSE4 construct, with GFP integrated at residue 83, was integrated at URA3, and the endogenous CSE4 was deleted (Chen et al., 2000). "
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    ABSTRACT: In many eukaryotes, the centromere is epigenetically specified and not strictly defined by sequence. In contrast, budding yeast has a specific 125 bp sequence required for kinetochore function. Despite the difference in centromere specification, budding yeast and multicellular eukaryotic centromeres contain a highly conserved histone H3 variant, CENP-A. The localization of budding yeast CENP-A, Cse4, requires the centromere DNA binding components, which are not conserved in multicellular eukaryotes. Here, we report that Cse4 localizes and functions at a synthetic kinetochore assembly site that lacks centromere sequence. The outer kinetochore Dam1-DASH and inner kinetochore CBF3 complexes are required for Cse4 localization to that site. Furthermore, the natural kinetochore also requires the outer kinetochore proteins for full Cse4 localization. Our results suggest that Cse4 localization at a functional kinetochore does not require the recognition of a specific DNA sequence by the CBF3 complex; rather, its localization depends on stable interactions among kinetochore proteins. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Dec 2014 · Cell Reports
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    • "Dam1 complex-coated beads follow growing or shrinking microtubule ends in vitro (Asbury et al., 2006). Importantly, artificially tethering the Dam1 complex to DNA is sufficient to segregate DNA in budding yeast (Kiermaier et al., 2009; Lacefield et al., 2009). The kinetochore localization of the Dam1 complex depends on its direct binding to the internal loop of Ndc80 (Maure et al., 2011), consistent with the notion that the Dam1 complex couples the movement of cargo to depolymerizing microtubules. "
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    ABSTRACT: Proper chromosome segregation during cell division is essential for proliferation, and this is facilitated by kinetochores, large protein complexes assembled on the centromeric region of the chromosomes. Although the sequences of centromeric DNA differ totally among organisms, many components of the kinetochores assembled on centromeres are very well conserved among eukaryotes. To define the identity of centromeres, centromere protein A (CENP-A), which is homologous to canonical histone H3, acts as a landmark for kinetochore assembly. Kinetochores mediate spindle-microtubule attachment and control the movement of chromosomes during mitosis and meiosis. To conduct faithful chromosome segregation, kinetochore assembly and microtubule attachment are elaborately regulated. Here we review the current understanding of the composition, assembly, functions and regulation of kinetochores revealed mainly through studies on fission and budding yeasts. Moreover, because recent cumulative evidence suggests the importance of the regulation of the orientation of kinetochore-microtubule attachment, which differs distinctly between mitosis and meiosis, we focus especially on the molecular mechanisms underlying this regulation.
    Full-text · Article · Oct 2013 · FEMS microbiology reviews
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    • "In fact, various groups have tried to create an artificial kinetochore by genetic engineering at a non-centromere region of a chromosome in recent years. This approach was first performed in budding yeast using plasmid DNA (Kiermaier et al. 2009; Lacefield et al. 2009). These experiments revealed that tethering of outer-kinetochore protein Dam1 is sufficient for generation of a functional kinetochore. "
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    ABSTRACT: The centromere is essential for accurate chromosome segregation during mitosis and meiosis to achieve transmission of genetic information to daughter cells. To facilitate accurate chromosome segregation, the centromere serves several specific functions, including microtubule binding, spindle-checkpoint control, and sister chromatid cohesion. The kinetochore is formed on the centromere to achieve these functions. To understand kinetochore structure and function, it is critical to identify the protein components of the kinetochore and characterize the functional properties of each component. Here, we review recent progress with regard to the molecular architecture of the kinetochore and discuss the future directions for centromere biology.
    Preview · Article · Jun 2012 · Chromosome Research
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