Article

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: 20.06). 09/2009; 11(9):1116-20. DOI: 10.1038/ncb1925
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
84 Views
 · 
0 Downloads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Chromosome Research 06/2012; 20(5):547-61. DOI:10.1007/s10577-012-9289-9 · 2.69 Impact Factor
  • Source
    • "Recently, a series of papers have illustrated the utility of using protein tethering to confer a genetic specification mechanism to centromeric regions. In yeast, a microtubule binding protein (Dam1) was tethered to a DNA-binding array and shown to promote segregation of an otherwise acentric plasmid (Kiermaier et al., 2009; Lacefield et al., 2009). Similarly , tethering of the human kinetochore proteins CENPC or CENPN was sufficient to assemble outer kinetochore proteins over an integrated repeat array (Gascoigne et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Plant genome engineering as a practical matter will require stable introduction of long and complex segments of DNA sequence into plant genomes. Here we show that it is possible to synthetically engineer and introduce centromere-sized satellite repeat arrays into maize. We designed a synthetic repeat monomer of 156 bp that contains five DNA-binding motifs (LacO, TetO, Gal4, LexA, and CENPB), and extended it into tandem arrays using an overlapping PCR method similar to that commonly used in gene synthesis. The PCR products were then directly transformed into maize using biolistic transformation. We identified three resulting insertion sites (arrayed binding sites), the longest of which is at least 1100 kb. The LacI DNA-binding module is sufficient to efficiently tether YFP to the arrayed binding sites. We conclude that synthetic repeats can be delivered into plant cells by omitting passage through Escherichia coli, that they generally insert into one locus, and that great lengths may be achieved. It is anticipated that these experimental approaches will be useful for future applications in artificial chromosome design.
    The Plant Journal 01/2012; 70(2):357-65. DOI:10.1111/j.1365-313X.2011.04867.x · 6.82 Impact Factor
  • Source
    • "Perhaps the centromere imposes geometry on the nascent kinetochore that is essential to build a functional kinetochore in this way. Topdown kinetochores do not require the inner kinetochore CBF3 complex (Lacefield et al. 2009), indicating that the major role for this complex is to act as a platform upon which to build the outer kinetochore. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulated interaction between kinetochores and the mitotic spindle is essential for the fidelity of chromosome segregation. Potentially deleterious attachments are corrected during prometaphase and metaphase. Correct attachments must persist during anaphase, when spindle-generated forces separate chromosomes to opposite poles. In yeast, the heterodecameric DASH complex plays a vital pole in maintaining this link. In vitro DASH forms both oligomeric patches and rings that can form load-bearing attachments with the tips of polymerising and depolymerising microtubules. In vivo, DASH localises primarily at the kinetochore, and has a role maintaining correct attachment between spindles and chromosomes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Recent work has begun to describe how DASH acts alongside other components of the outer kinetochore to create a dynamic, regulated kinetochore-microtubule interface. Here, we review some of the key experiments into DASH function and discuss their implications for the nature of kinetochore-microtubule attachments in yeast and other organisms.
    Chromosome Research 04/2011; 19(3):393-407. DOI:10.1007/s10577-011-9185-8 · 2.69 Impact Factor
Show more

Preview

Download
0 Downloads
Available from