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Insights into kinetochore-DNA interactions from the structure of Cep3Delta.

Purvis A, Singleton MR - EMBO Rep. (2007)

Bottom Line: The dimer has a large central channel that is sufficient to accommodate duplex B-form DNA.The zinc-finger domains emerge at the edges of the channel, and could bind to the DNA in a pseudo-symmetrical manner at degenerate half-sites in the centromeric sequence.We propose a mechanism for the modulation of DNA affinity by an acidic activator domain, which could be applicable to a wider family of transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Macromolecular Structure and Function Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, UK.

ABSTRACT
The CBF3 complex is an essential core component of the budding yeast kinetochore and is required for the centromeric localization of all other kinetochore proteins. We determined the crystal structure of a large section of the protein Cep3 from CBF3, which is the only component with obvious DNA-binding motifs. The protein adopts a roughly bilobal shape, with an extended dimerization interface. The dimer has a large central channel that is sufficient to accommodate duplex B-form DNA. The zinc-finger domains emerge at the edges of the channel, and could bind to the DNA in a pseudo-symmetrical manner at degenerate half-sites in the centromeric sequence. We propose a mechanism for the modulation of DNA affinity by an acidic activator domain, which could be applicable to a wider family of transcription factors.

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Structure of the budding yeast centromere and organization of the CBF3 complex. (A) Schematic diagram of the budding yeast centromere element showing the location and scale of the CDE elements, as well as the consensus DNA composition in CDEIII. (B) Diagram of the four components of the CBF3 complex on DNA. (C) Schematic of the Cep3 sequence against Gal4 showing the locations of the conserved DNA-binding domain (yellow) and acidic patch (purple). The orange bar indicates the fragment described in this study. (D) Sequence alignment of members of the zinc-cluster domains from members of the Gal4-family of transcription factors against the amino-terminal of Cep3. The secondary structure elements are shown above the alignment, and totally conserved residues are shown in red.
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f1: Structure of the budding yeast centromere and organization of the CBF3 complex. (A) Schematic diagram of the budding yeast centromere element showing the location and scale of the CDE elements, as well as the consensus DNA composition in CDEIII. (B) Diagram of the four components of the CBF3 complex on DNA. (C) Schematic of the Cep3 sequence against Gal4 showing the locations of the conserved DNA-binding domain (yellow) and acidic patch (purple). The orange bar indicates the fragment described in this study. (D) Sequence alignment of members of the zinc-cluster domains from members of the Gal4-family of transcription factors against the amino-terminal of Cep3. The secondary structure elements are shown above the alignment, and totally conserved residues are shown in red.

Mentions: The kinetochore is a large proteinaceous complex that is responsible for linking chromatids to spindle microtubules during mitosis (Westermann et al, 2007). It transmits force from the mitotic spindle to the chromatids, generates motion through associated motor proteins and creates a checkpoint signal to prevent premature entry into anaphase. The kinetochore shows a high degree of functional conservation between species, although there is considerable sequence variability at the protein level (Meraldi et al, 2006). More than 65 proteins have been associated with the yeast kinetochore, of which many have been shown to form relatively stable sub-complexes (De Wulf et al, 2003). These associate in a hierarchical and tightly controlled manner on a specialized region of the chromosome known as the centromere. Despite recent advances (Wei et al, 2005) there are still relatively little high-resolution structural data available for kinetochore proteins. One area of interest is how the inner layer of the kinetochore binds to the appropriate centromeric DNA sequence. In budding yeast, the minimal essential DNA (CEN) required for kinetochore formation is only approximately 125 bp in length and has a defined sequence, which is both necessary and sufficient for kinetochore assembly (Clarke & Carbon, 1980). The centromere consists of three elements: CDEI, CDEII and CDEIII (Fitzgerald-Hayes et al, 1982; Fig 1A). The central CDEII element (78–86 bp) has a degenerate A/T-rich sequence, whereas CDEI (8 bp) and CDEIII (25 bp) are highly conserved. Deletions in either CDEI or CDEII degrade the fidelity of chromosome segregation but do not prevent it (Carbon & Clarke, 1984); by contrast, deletion of CDEIII entirely abolishes centromere activity (Ng & Carbon, 1987). The sequence of the CDEIII element shows partial dyad symmetry and contains a totally conserved CCG motif, in which point mutations cause a complete loss of CEN function (Jehn et al, 1991). Various proteins have been localized to the different CDE elements. The CDEIII element is bound by the CBF3 complex (Lechner & Carbon, 1991). This contains four proteins: Cep3 (Lechner, 1994; Strunnikov et al, 1995), Ctf13 (Doheny et al, 1993), Ndc10 (Goh & Kilmartin, 1993; Jiang et al, 1993) and Skp1 (Connelly & Hieter, 1996; Stemmann & Lechner, 1996). All four proteins are essential and formation of the complex is necessary for recruitment of all other kinetochore components (Sorger et al, 1994; He et al, 2001).


Insights into kinetochore-DNA interactions from the structure of Cep3Delta.

Purvis A, Singleton MR - EMBO Rep. (2007)

Structure of the budding yeast centromere and organization of the CBF3 complex. (A) Schematic diagram of the budding yeast centromere element showing the location and scale of the CDE elements, as well as the consensus DNA composition in CDEIII. (B) Diagram of the four components of the CBF3 complex on DNA. (C) Schematic of the Cep3 sequence against Gal4 showing the locations of the conserved DNA-binding domain (yellow) and acidic patch (purple). The orange bar indicates the fragment described in this study. (D) Sequence alignment of members of the zinc-cluster domains from members of the Gal4-family of transcription factors against the amino-terminal of Cep3. The secondary structure elements are shown above the alignment, and totally conserved residues are shown in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2246632&req=5

f1: Structure of the budding yeast centromere and organization of the CBF3 complex. (A) Schematic diagram of the budding yeast centromere element showing the location and scale of the CDE elements, as well as the consensus DNA composition in CDEIII. (B) Diagram of the four components of the CBF3 complex on DNA. (C) Schematic of the Cep3 sequence against Gal4 showing the locations of the conserved DNA-binding domain (yellow) and acidic patch (purple). The orange bar indicates the fragment described in this study. (D) Sequence alignment of members of the zinc-cluster domains from members of the Gal4-family of transcription factors against the amino-terminal of Cep3. The secondary structure elements are shown above the alignment, and totally conserved residues are shown in red.
Mentions: The kinetochore is a large proteinaceous complex that is responsible for linking chromatids to spindle microtubules during mitosis (Westermann et al, 2007). It transmits force from the mitotic spindle to the chromatids, generates motion through associated motor proteins and creates a checkpoint signal to prevent premature entry into anaphase. The kinetochore shows a high degree of functional conservation between species, although there is considerable sequence variability at the protein level (Meraldi et al, 2006). More than 65 proteins have been associated with the yeast kinetochore, of which many have been shown to form relatively stable sub-complexes (De Wulf et al, 2003). These associate in a hierarchical and tightly controlled manner on a specialized region of the chromosome known as the centromere. Despite recent advances (Wei et al, 2005) there are still relatively little high-resolution structural data available for kinetochore proteins. One area of interest is how the inner layer of the kinetochore binds to the appropriate centromeric DNA sequence. In budding yeast, the minimal essential DNA (CEN) required for kinetochore formation is only approximately 125 bp in length and has a defined sequence, which is both necessary and sufficient for kinetochore assembly (Clarke & Carbon, 1980). The centromere consists of three elements: CDEI, CDEII and CDEIII (Fitzgerald-Hayes et al, 1982; Fig 1A). The central CDEII element (78–86 bp) has a degenerate A/T-rich sequence, whereas CDEI (8 bp) and CDEIII (25 bp) are highly conserved. Deletions in either CDEI or CDEII degrade the fidelity of chromosome segregation but do not prevent it (Carbon & Clarke, 1984); by contrast, deletion of CDEIII entirely abolishes centromere activity (Ng & Carbon, 1987). The sequence of the CDEIII element shows partial dyad symmetry and contains a totally conserved CCG motif, in which point mutations cause a complete loss of CEN function (Jehn et al, 1991). Various proteins have been localized to the different CDE elements. The CDEIII element is bound by the CBF3 complex (Lechner & Carbon, 1991). This contains four proteins: Cep3 (Lechner, 1994; Strunnikov et al, 1995), Ctf13 (Doheny et al, 1993), Ndc10 (Goh & Kilmartin, 1993; Jiang et al, 1993) and Skp1 (Connelly & Hieter, 1996; Stemmann & Lechner, 1996). All four proteins are essential and formation of the complex is necessary for recruitment of all other kinetochore components (Sorger et al, 1994; He et al, 2001).

Bottom Line: The dimer has a large central channel that is sufficient to accommodate duplex B-form DNA.The zinc-finger domains emerge at the edges of the channel, and could bind to the DNA in a pseudo-symmetrical manner at degenerate half-sites in the centromeric sequence.We propose a mechanism for the modulation of DNA affinity by an acidic activator domain, which could be applicable to a wider family of transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Macromolecular Structure and Function Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London, UK.

ABSTRACT
The CBF3 complex is an essential core component of the budding yeast kinetochore and is required for the centromeric localization of all other kinetochore proteins. We determined the crystal structure of a large section of the protein Cep3 from CBF3, which is the only component with obvious DNA-binding motifs. The protein adopts a roughly bilobal shape, with an extended dimerization interface. The dimer has a large central channel that is sufficient to accommodate duplex B-form DNA. The zinc-finger domains emerge at the edges of the channel, and could bind to the DNA in a pseudo-symmetrical manner at degenerate half-sites in the centromeric sequence. We propose a mechanism for the modulation of DNA affinity by an acidic activator domain, which could be applicable to a wider family of transcription factors.

Show MeSH