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

1.钳位装载复合物与DNA聚合酶的功能相互作用钳位装载复合物之一，Ctf 18-RFC，由染色体凝聚因子Ctf 18，Dccl和Ctf 8以及RFC的四个小亚基组成，作为第二个PCNA装载物。我们确定Pol eta为Ctf 18-RFC而非RFC刺激的DNA聚合酶活性。Ctf 18-RFC单独刺激Pol eta，并且PCNA的添加协同地增加该刺激。此外，Ctf 18-RFC与pol eta发生物理相互作用。此外，我们还观察到RFC与pol eta的类似的物理相互作用，但其对DNA合成活性的影响与Ctf 18-RFC不同。我们提出这种新的装载剂-DNA聚合酶相互作用可以改变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loader复合物FLAG-RFC、FLAG-（Rad 17-RFC）、FLAG-（Ctf 18-RFC）的人细胞系，我们鉴定出Pol-1和RPA是常见的loader结合蛋白。对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和特定的亚基与这些蛋白质结合，这表明它们在复制叉处维持功能性蛋白质组装的作用。WRNIP 1蛋白的功能我们纯化了RFC相关蛋白之一WRNIP 1，它已被鉴定为Werner DNA解旋酶结合蛋白。该蛋白质形成自寡聚复合物并表现出ATP酶活性。它还与复制型DNA聚合酶δ特异性相互作用并刺激其活性，表明其在Pol δ调节DNA合成中的作用。

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.