Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae
Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae
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DOI：
10.1128/mcb.7.12.4522-4534.1987
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发表时间：
1987-12
影响因子：
5.3
通讯作者：
NG Ray;And;John Carbon
中科院分区：
文献类型：
--
作者：
NG Ray;And;John Carbon
酿酒酵母染色体上的着丝粒含有约140个碱基对（bp）的DNA。功能性着丝粒（CEN）区域包含三个重要的序列元件（I，PuTCACPuTG; II，78至86 bp的高AT DNA;和III，具有内部双侧对称性的保守的25 bp序列）。元件III区的各种点突变或缺失对CEN在体内的功能具有深远的影响，表明该DNA区域是关键的蛋白结合位点。这已经通过使用两种体外测定来检测酵母蛋白与含有野生型或突变改变的CEN 3序列的DNA片段的结合而得到证实。使用核酸外切酶III保护测定来证明蛋白质与CEN 3的元件III区域的特异性结合。此外，使用凝胶DNA片段迁移率变动测定来表征结合反应参数。在体内发挥功能的序列元件III突变也阻止了体外测定中蛋白质的结合。迁移率变动分析表明，含有序列元件III的双链DNA在不存在序列元件I和II的情况下有效地结合蛋白质，尽管后者的序列对于体内最佳CEN功能是必需的。
Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.