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一种人工合成的人类激酶可以控制出芽酵母中的细胞周期进程。

A synthetic human kinase can control cell cycle progression in budding yeast.

机构信息

Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

出版信息

G3 (Bethesda). 2011 Sep;1(4):317-25. doi: 10.1534/g3.111.000430. Epub 2011 Sep 1.

Abstract

The DDK kinase complex, composed of Cdc7 and Dbf4, is required for S-phase progression. The two component proteins show different degrees of sequence conservation between human and yeast. Here, we determine that Saccharomyces cerevisiae bearing human CDC7 and DBF4 grows comparably to cells with yeast DDK under standard growth conditions. HsDrf1 (a second human Dbf4-like protein) does not support growth, suggesting that HsDbf4 is the true ortholog of ScDbf4. Both human subunits are required to complement yeast cdc7Δ or dbf4Δ due to the inability of human Cdc7 or Dbf4 to interact with the corresponding yeast protein. Flow cytometry indicates normal cell cycle progression for yeast containing human DDK. However, yeast containing human DDK is sensitive to long-term exposure to hydroxyurea and fails to sporulate, suggesting that human DDK substitutes for some, but not all, of yeast DDK's functions. We mapped the region of Cdc7 required for species-specific function of DDK to the C-terminus of Cdc7 by substituting the yeast C-terminal 55 amino acid residues in place of the equivalent human residues. The resulting hybrid protein supported growth of a cdc7Δ strain only in the presence of ScDBF4. The strain supported by the hybrid CDC7 was not sensitive to HU and formed tetrads. Together, our data indicate that DDK's targeting of its essential substrate is conserved between species, whereas the interactions within DDK are species specific.

摘要

DDK 激酶复合物由 Cdc7 和 Dbf4 组成,是 S 期进展所必需的。这两种成分蛋白在人类和酵母之间表现出不同程度的序列保守性。在这里,我们确定了携带人类 CDC7 和 DBF4 的酿酒酵母在标准生长条件下的生长情况与具有酵母 DDK 的细胞相当。HsDrf1(第二种人类 Dbf4 样蛋白)不能支持生长,这表明 HsDbf4 是 ScDbf4 的真正同源物。由于人类 Cdc7 或 Dbf4 无法与相应的酵母蛋白相互作用,因此需要这两种人类亚基来补充酵母 cdc7Δ 或 dbf4Δ。流式细胞术表明,含有人类 DDK 的酵母细胞的细胞周期进程正常。然而,含有人类 DDK 的酵母对长期暴露于羟基脲敏感,并且无法形成孢子,这表明人类 DDK 替代了一些但不是所有的酵母 DDK 功能。我们通过用酵母 C 端的 55 个氨基酸残基取代人类 C 端的等效残基,将 DDK 种特异性功能所需的 Cdc7 区域定位到 Cdc7 的 C 末端。产生的杂交蛋白仅在存在 ScDBF4 的情况下支持 cdc7Δ 菌株的生长。由杂交 CDC7 支持的菌株对 HU 不敏感并且形成四分体。总之,我们的数据表明,DDK 对其必需底物的靶向在物种间是保守的,而 DDK 内的相互作用是物种特异性的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fa7/3276143/aaaa8bac85b3/317f1.jpg

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