The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-1ike domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.
A universal mark of centromeric chromatin is its packaging by a variant of histone H3 known as centromeric H3 (CenH3). The mechanism by which CenH3s are incorporated specifically into centromere DNA or the specialized function they serve there is not known. In a genetic approach to identify factors involved in CenH3 deposition, we screened for dosage suppressors of a temperaturesensitive cse4 allele in Saccharomyces cerevisiae (Cse4 is the S. cerevisiae CenH3). Independent screens yielded ORF YDL139C, which we named SCM3. Dosage suppression by SCM3 was specific for alleles affecting the histone fold domain of Cse4. Copurification and two-hybrid studies showed that Scm3 and Cse4 interact in vivo, and chromatin immunoprecipitation revealed that Scm3, like Cse4, is found associated with centromere DNA. Scm3 contains two essential protein domains, a Leu-rich nuclear export signal and a heptad repeat domain that is widely conserved in fungi. A conditional scm3 allele was generated to allow us to deplete Scm3. Upon Scm3 depletion, cells undergo a Mad2-dependent G 2/M arrest, and centromere localization of Cse4 is perturbed. We suggest that S. cerevisiae Scm3 defines a previously undescribed family of fungal kinetochore proteins important for CenH3 localization.centromeric H3 ͉ chromosome segregation ͉ kinetochore ͉ yeast ͉ CENP-A
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