Purification of the Yeast Centromere Binding Protein CP1 and a Mutational Analysis of Its Binding Site

• Published in: The Journal of biological chemistry 1989-06, Vol.264 (18), p.10843-10850
• Main Authors: Baker, R E, Fitzgerald-Hayes, M, O'Brien, T C
• Format: Article
• Language: English
• Subjects: 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Analytical, structural and metabolic biochemistry; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Centromere - metabolism; Chromosomes - metabolism; DNA, Fungal - genetics; DNA, Fungal - metabolism; DNA-Binding Proteins - isolation & purification; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular Sequence Data; Plasmids; Protein Binding; Proteins; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)81697-0

CP1 is a yeast protein which binds to the highly conserved DNA element I (CDEI) of yeast centromeres. We have purified CPl to near homogeneity; it is comprised of a single polypeptide of molecular weight 58,400. When bound to yeast CEN3 DNA, CPl protects a 12–15-base pair region centered over CDEI. Methylation interference experiments show that methylations of residues located outside of the 8-base pair CDEI sequence have no detectable effect on CP1 binding, suggesting that the DNA sequences important for CP1 recognition are confined to the CDEI octanucleotide. The equilibrium constant for CP1 binding to CEN3 DNA is relatively low, 3 × 108M−1. Using a novel method to determine relative DNA binding constants, we analyzed the effect of CDEI mutations on CP1 binding. A C to T point mutation at position 5 (CO1) reduces the equilibrium constant about 35-fold, while the insertion of an additional T at this position (CAT) reduces the equilibrium constant 1,400-fold. The effect of these mutations on mitotic centromere function in vivo was assessed using a plasmid stability assay. While the CO1 mutation had a slight effect, the CAT mutation significantly impaired function, implying that CP1 binding is required for the optimal mitotic function of yeast centromeres.