Title

Purification of the yeast centromere binding protein CP1 and a mutational analysis of its binding site

UMMS Affiliation

Department of Molecular Genetics and Microbiology

Date

6-25-1989

Document Type

Article

Subjects

3',5'-Cyclic-AMP Phosphodiesterases; Base Sequence; Centromere; Chromosomes; DNA, Fungal; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Kinetics; Molecular Sequence Data; Plasmids; Protein Binding; Restriction Mapping; Saccharomyces cerevisiae

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

CP1 is a yeast protein which binds to the highly conserved DNA element I (CDEI) of yeast centromeres. We have purified CP1 to near homogeneity; it is comprised of a single polypeptide of molecular weight 58,400. When bound to yeast CEN3 DNA, CP1 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 x 10(8) M-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.

Rights and Permissions

Citation: J Biol Chem. 1989 Jun 25;264(18):10843-50.

Related Resources

Link to Article in PubMed

Journal Title

The Journal of biological chemistry

PubMed ID

2543684