Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3

Cse4p is a variant of histone H3 that has an essential role in chromosome s egregation and centromere chromatin structure in budding yeast. Cse3p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere prot ein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammal s, respectively. In order to identify regions of Cse3p that distinguish it from H3 and confer centromere function, a systematic site-directed mutation al analysis was performed. Nested deletions of the Cse3p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p ami no acids that differ between Cse3p and H3 to the analogous H3 residues. Ext ensive substitution of contiguous Cse3p residues with H3 counterparts resul ted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated r ates of mitotic chromosome loss. The results indicate that residues critica l for wild-type Cse4p function and high-fidelity chromosome transmission ar e distributed across the entire histone-fold domain. Our findings are discu ssed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A.