Researches on functional link between DNA replication and chromosomal cohesion through PCNA clamp

1.钳位加载器复合体与DNA聚合酶的功能相互作用Ctf18-RFC是一种由染色体结合因子Ctf18、Dcc1、Ctf8和RFC的四个小亚基组成的Ctf18-RFC，其功能是作为第二个增殖细胞核抗原加载器。我们鉴定Pol ETA是由Ctf18-RFC刺激的DNA聚合酶活性，而不是RFC刺激的DNA聚合酶活性。Ctf18-RFC单独刺激Pol ETA，而增殖细胞核抗原的加入可协同增强这种刺激作用。此外，Ctf18-RFC与PolETA发生物理相互作用。此外，我们观察到RFC与PolETA具有相似的物理相互作用，但对其DNA合成活性的影响不同于Ctf18-RFC。我们认为，新的加载器-DNA聚合酶相互作用可以切换DNA复制叉的活性，以适应不同的模板结构。利用异位表达标记的加载器复合体FLAG-RFC、FLAG-(Rad17-RFC)、FLAG-(Ctf18-RFC)的人细胞系，我们鉴定了Pol epsilon和RPA是常见的加载器结合蛋白。与重组蛋白的进一步研究表明，它们之间的相互作用是直接和特异的。所有的加载子都可以通过它们共同的小亚基，也可以通过特定的亚基与这些蛋白质结合，这暗示了在复制分叉处维持蛋白质功能组装的作用。WRNIP1蛋白的功能我们纯化了RFC相关蛋白之一WRNIP1，该蛋白已被鉴定为Werner DNA解旋酶结合蛋白。这种蛋白质形成一个自我寡聚的复合体，并显示出ATPase活性。它还与复制的DNA聚合酶Delta特异地相互作用并刺激其活性，表明它在Pol Delta调节DNA合成中发挥作用。

1. Functional interaction of clamp loader complexes with DNA polymerase etaOne of clamp loader complexes, Ctf18-RFC, which consists of the chromosome cohesion factors Ctf18, Dccl, and Ctf8 and four small subunits of RFC, functions as a second PCNA loader. We identified Pol eta as a DNA polymerase activity stimulated by Ctf18-RFC but not by RFC. Ctf18-RFC stimulated Pol eta on its own, and the addition of PCNA cooperatively increased that stimulation. Furthermore, Ctf18-RFC interacted physically with pol eta. In addition, we observed the similar physical interaction of RFC with pol eta, but affecting its DNA synthesis activity differently from Ctf18-RFC. We propose that the novel loader-DNA polymerase interactions can switch the activity of a DNA replication fork to adapt to various template structures.2. Molecular interaction between loader complexes and replication fork componentsUsing human cell lines ectopically expressing tagged loader complexes, FLAG-RFC, FLAG-(Rad17-RFC), FLAG-(Ctf18-RFC), we have identified Pol epsilon and RPA as common loader binding proteins. Further studies with recombinant proteins have indicated that their interactions are direct and specific. All loaders can associate with these proteins through their common small subunits, and also through specific subunits, suggesting roles to maintain functional protein assemblies at the replication fork.3. Functions of WRNIP1 proteinWe purified one of RFC-related proteins, WRNIP1, which has been identified as a Werner DNA helicase binding protein. This protein forms a self-oligomerized complex and exhibits ATPase activity. It also interacts specifically with replicative DNA polymerase delta and stimulates the activity, suggesting its role in regulation of DNA synthesis by Pol delta.