Abstract
In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans ~56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms. It spans ~80 bp of DNA. CBF3 makes sequence-specific and - nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3- centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1383-1396 |
| Number of pages | 14 |
| Journal | Journal of Cell Biology |
| Volume | 139 |
| Issue number | 6 |
| DOIs | |
| State | Published - Dec 15 1997 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Cell Biology
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Probing the architecture of a simple kinetochore using DNA-protein crosslinking. / Espelin, Christopher W.; Kaplan, Kenneth B.; Sorger, Peter K.
In: Journal of Cell Biology, Vol. 139, No. 6, 15.12.1997, p. 1383-1396.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Probing the architecture of a simple kinetochore using DNA-protein crosslinking
AU - Espelin, Christopher W.
AU - Kaplan, Kenneth B.
AU - Sorger, Peter K.
PY - 1997/12/15
Y1 - 1997/12/15
N2 - In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans ~56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms. It spans ~80 bp of DNA. CBF3 makes sequence-specific and - nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3- centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.
AB - In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans ~56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms. It spans ~80 bp of DNA. CBF3 makes sequence-specific and - nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3- centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.
UR - http://www.scopus.com/inward/record.url?scp=0031451181&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0031451181&partnerID=8YFLogxK
U2 - 10.1083/jcb.139.6.1383
DO - 10.1083/jcb.139.6.1383
M3 - Article
C2 - 9396745
AN - SCOPUS:0031451181
VL - 139
SP - 1383
EP - 1396
JO - Journal of Cell Biology
JF - Journal of Cell Biology
SN - 0021-9525
IS - 6
ER -