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Analysis of structural determinants involved in yeast Cse4p -CEN DNA interactions: Implications for the chromatin structure of eukaryotic centromeres
Cse4p is a centromere-specific chromatin protein with a histone-fold domain that is greater than 60% identical to histone H3 and the mammalian centromere protein, CENP-A. Cse4p has similar biochemical properties to H3 and is believed to replace H3 in centromere-specific nucleosomes in yeast. To identify residues in the histone-fold domain of Cse4p that function in specifying centromere structure and function, amino adds that differ between Cse4p and H3 were systematically changed to analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The severity of the phenotypes exhibited by the histone-fold domain mutants correlates directly with the number of putative DNA contact sites changed or alterations in regions implicated in histone interactions. These results indicate that the histone-fold domain of Cse4p functions through a cooperative mechanism in which residues throughout the histone-fold domain recognize centromere DNA. To investigate the relationship between the histone-fold domain of Cse4p and centromere DNA, chromosome loss rates were measured in double mutants carrying both a mutation in Cse4p and chromosomes with mutant centromere DNA. The Cse4p mutants had conserved changes in which analogously positioned residues in H3 were substituted for Cse4p residues at sites in the histone-fold domain that are in close proximity to the DNA. Mutations throughout the histone-fold domain caused significant increases in chromosome loss rates of chromosomes carrying conserved centromere DNA elements CDE I and II but showed no effect with CDE III mutant centromeres. This genetic evidence strongly supports direct interactions between Cse4p and CDE I and CDE II centromere DNA elements. The results are discussed in the context of the known structure of H3 and models are proposed which best describe the path of the centromere DNA around a Cse4p variant nucleosome that relies on critical contacts between CDE I and II, but not CDE III.
Keith, Kevin C, "Analysis of structural determinants involved in yeast Cse4p -CEN DNA interactions: Implications for the chromatin structure of eukaryotic centromeres" (1999). Doctoral Dissertations Available from Proquest. AAI9950170.