Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein.
Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein.
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DOI：
10.1083/jcb.152.5.985
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发表时间：
2001-03-05
影响因子：
7.8
通讯作者：
Yan, H
中科院分区：
文献类型：
--
作者：
Chen, C Y;Graham, J;Yan, H
高等真核细胞中的DNA复制发生在大量被称为复制焦点的离散位点上。我们先前已经纯化了一种蛋白，聚焦形成活性1 (FFA-1)，它参与了爪蟾卵提取物中假定的复制前聚焦的组装。FFA-1是Werner综合征基因产物（WRN）的同源物，WRN是RecQ解旋酶家族的成员。在本文中，我们发现FFA-1与DNA合成位点和单链DNA结合蛋白复制蛋白A （RPA）共定位于鸡蛋提取物重建的细胞核中。此外，我们发现两种谷胱甘肽s -转移酶FFA-1融合蛋白可以以显性负向方式抑制DNA复制。主要的负面影响与融合蛋白结合到复制病灶形成“杂交病灶”有关，杂交病灶无法参与DNA复制。在生化水平上，RPA可与FFA-1相互作用，特异性刺激其DNA解旋酶活性。然而，在显性负突变蛋白的存在下，刺激被阻止。这些结果为FFA-1在DNA复制中的重要作用提供了第一个直接的生化证据。
DNA replication in higher eukaryotic cells occurs at a large number of discrete sites called replication foci. We have previously purified a protein, focus-forming activity 1 (FFA-1), which is involved in the assembly of putative prereplication foci in Xenopus egg extracts. FFA-1 is the orthologue of the Werner syndrome gene product (WRN), a member of the RecQ helicase family. In this paper we show that FFA-1 colocalizes with sites of DNA synthesis and the single-stranded DNA binding protein, replication protein A (RPA), in nuclei reconstituted in the egg extract. In addition, we show that two glutathione S-transferase FFA-1 fusion proteins can inhibit DNA replication in a dominant negative manner. The dominant negative effect correlates with the incorporation of the fusion proteins into replication foci to form “hybrid foci,” which are unable to engage in DNA replication. At the biochemical level, RPA can interact with FFA-1 and specifically stimulates its DNA helicase activity. However, in the presence of the dominant negative mutant proteins, the stimulation is prevented. These results provide the first direct biochemical evidence of an important role for FFA-1 in DNA replication.