AN ESSENTIAL YEAST PROTEIN, CBF5P, BINDS IN-VITRO TO CENTROMERES AND MICROTUBULES
AN ESSENTIAL YEAST PROTEIN, CBF5P, BINDS IN-VITRO TO CENTROMERES AND MICROTUBULES
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DOI：
10.1128/mcb.13.8.4884
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发表时间：
1993-08-01
影响因子：
5.3
通讯作者：
CARBON, J
中科院分区：
文献类型：
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作者：
JIANG, WD;MIDDLETON, K;CARBON, J
酵母着丝粒DNA(CEN)亲和柱层析已用于从酵母染色质提取物中纯化几种可能的着丝粒和着丝粒蛋白。酵母单基因(CBF5)是一种重要的低亲和力着丝粒结合蛋白(p64‘/CBF5p)，已被克隆并被证明是酿酒酵母生存所必需的。CBF5指定一个55 kDa的高电荷蛋白，在C末端附近包含一个重复的KKD/E序列结构域，类似于微管相关蛋白1A和1B中已知的微管结合结构域。通过在细菌细胞中过表达获得的CBF5p在体外与微管结合，而缺乏(KKD/E)n结构域的C末端缺失蛋白不能结合微管。分离含有C端截短的CBF5基因的酵母细胞，产生只包含三个KKD/E重复序列的CBF5p，延迟细胞周期的G2/M阶段的复制基因组，而CBF5p的缺失阻止了G1/S中的大多数细胞。酿酒酵母中CBF5p的过量生产补充了该基因(CBF2)的温度敏感性突变，该基因指定了高亲和力CEN DNA结合因子CBF3的110 kDa亚基，表明CBF5p和CBF3在体内相互作用。第二个低亲和力着丝粒结合因子被鉴定为拓扑异构酶II。
Yeast centromere DNA (CEN) affinity column chromatography has been used to purify several putative centromere and kinetochore proteins from yeast chromatin extracts. The single yeast gene (CBF5) specifying one of the major low-affinity centromere-binding proteins (p64'/CBF5p) has been cloned and shown to be essential for viability of Saccharomyces cerevisiae. CBF5 specifies a 55-kDa highly charged protein that contains a repeating KKD/E sequence domain near the C terminus, similar to known microtubule-binding domains in microtubule-associated proteins 1A and 1B. CBF5p, obtained by overexpression in bacterial cells, binds microtubules in vitro, whereas C-terminal deleted proteins lacking the (KKD/E)n domain do not. Dividing yeast cells containing a C-terminal truncated CBF5 gene, producing CBF5p containing only three copies of the KKD/E repeat, delay with replicated genomes at the G2/M phase of the cell cycle, while depletion of CBF5p arrests most cells in G1/S. Overproduction of CBF5p in S. cerevisiae complements a temperature sensitivity mutation in the gene (CBF2) specifying the 110-kDa subunit of the high-affinity CEN DNA-binding factor CBF3, suggesting in vivo interaction of CBF5p and CBF3. A second low-affinity centromere-binding factor has been identified as topoisomerase II.