MUTATIONAL AND INVITRO PROTEIN-BINDING STUDIES ON CENTROMERE DNA FROM SACCHAROMYCES-CEREVISIAE
MUTATIONAL AND INVITRO PROTEIN-BINDING STUDIES ON CENTROMERE DNA FROM SACCHAROMYCES-CEREVISIAE
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DOI：
10.1128/mcb.7.12.4522
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发表时间：
1987-12-01
影响因子：
5.3
通讯作者：
CARBON, J
中科院分区：
文献类型：
--
作者：
NG, R;CARBON, J
酿酒酵母染色体上的着丝粒包含大约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到86bp的高AT含量DNA；以及III，一个具有内部双侧对称的保守的25bp序列）。元件III区域的各种点突变或缺失对体内CEN功能有深远影响，表明该DNA区域是一个关键的蛋白质结合位点。这已通过两种体外检测方法得到证实，这些方法用于检测酵母蛋白质与含有野生型或突变改变的CEN3序列的DNA片段的结合。使用了外切核酸酶III蛋白质检测方法来证明蛋白质与CEN3的元件III区域的特异性结合。此外，还使用了凝胶DNA片段迁移率变动分析来表征结合反应参数。在体内使CEN功能失活的序列元件III突变在体外检测中也阻止蛋白质结合。迁移率变动分析表明，含有序列元件III的双链DNA在没有序列元件I和II的情况下能有效地结合蛋白质，尽管后两个序列对于体内最佳CEN功能是必不可少的。
Centromeres on chromosomes in the yeast Saccharomyces cervisiae contain approximately 140 base pairs (bp) of DNA. The functional centrometer (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 protein 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.