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Molecular and Cellular Biology, September 2000, p. 7037-7048, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

The N Terminus of the Centromere H3-Like Protein Cse4p Performs an Essential Function Distinct from That of the Histone Fold Domain

Yinhuai Chen,¹ Richard E. Baker,² Kevin C. Keith,^1, Kendra Harris,² Sam Stoler,¹ and Molly Fitzgerald-Hayes^1,*

Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts at Amherst, Amherst, Massachusetts 01003,¹ and Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655²

Received 29 February 2000/Returned for modification 4 April 2000/Accepted 30 May 2000

Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation.

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^* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts at Amherst, Amherst, MA 01003. Phone: (413) 545-0235. Fax: (413) 545-3291. E-mail: mollyfh{at}biochem.umass.edu.

Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637.

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Molecular and Cellular Biology, September 2000, p. 7037-7048, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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