Lei X H, Shen X, Xu X Q, Bernstein H S
Department of Pediatrics, Cardiovascular Research Institute and Cancer Center, University of California, San Francisco, Box 0130, San Francisco, California 94143-0130, USA.
J Cell Sci. 2000 Dec;113 Pt 24:4523-31. doi: 10.1242/jcs.113.24.4523.
G(2)/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved. We recently identified human Cdc5, a positive regulator of G(2)/M in mammalian cells. We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G(2)/M through transcriptional activation. Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members. In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding. We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus. We show that this DNA-protein interaction is capable of activating transcription. We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5. Several of these contained sequences similar to the binding site. We demonstrate that these bind human Cdc5 with similar specificity and affinity. These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome. This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.
G(2)/M期转换需要多种基因产物的协同表达，但对于其中涉及的转录调节因子却知之甚少。我们最近鉴定出人类Cdc5，它是哺乳动物细胞中G(2)/M期的正向调节因子。我们还证明其羧基末端存在一个潜在的激活结构域，这表明人类Cdc5通过转录激活来调节G(2)/M期。尽管存在一个DNA结合结构域，但其他人的研究未能确定Cdc5家族成员的优先结合位点。此外，最近在几种生物体中发现Cdc5与剪接体有关联，这表明它可能不是通过DNA结合发挥作用。我们现在报告鉴定出一个12bp的序列，人类Cdc5通过其氨基末端与之特异性且高亲和力地结合。我们表明这种DNA-蛋白质相互作用能够激活转录。我们还利用酵母中的筛选系统来鉴定与人类Cdc5相互作用的人类基因组片段。其中几个片段包含与结合位点相似的序列。我们证明这些片段以相似的特异性和亲和力结合人类Cdc5。这些实验提供了首个证据，表明Cdc5家族成员可作为位点特异性DNA结合蛋白，并且人类Cdc5可能与人类基因组中特定的、低丰度序列相互作用。这增加了Cdc5蛋白可能参与调控细胞分裂所需的多个过程的可能性。
