Baker R E, Fitzgerald-Hayes M, O'Brien T C
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
J Biol Chem. 1989 Jun 25;264(18):10843-50.
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.
CP1是一种酵母蛋白，它与酵母着丝粒的高度保守DNA元件I（CDEI）结合。我们已将CP1纯化至近乎同质；它由一条分子量为58,400的单一多肽组成。当与酵母CEN3 DNA结合时，CP1保护以CDEI为中心的12 - 15个碱基对的区域。甲基化干扰实验表明，位于8个碱基对的CDEI序列之外的残基甲基化对CP1结合没有可检测到的影响，这表明对CP1识别重要的DNA序列局限于CDEI八聚体核苷酸。CP1与CEN3 DNA结合的平衡常数相对较低，为3×10⁸ M⁻¹。使用一种新颖的方法来确定相对DNA结合常数，我们分析了CDEI突变对CP1结合的影响。第5位的C到T点突变（CO1）使平衡常数降低约35倍，而在此位置额外插入一个T（CAT）使平衡常数降低1400倍。使用质粒稳定性测定法评估了这些突变对体内有丝分裂着丝粒功能的影响。虽然CO1突变有轻微影响，但CAT突变显著损害功能，这意味着CP1结合是酵母着丝粒最佳有丝分裂功能所必需的。
