Studies on interfaces between DNA replication and DNA damage responses through multiple clamp and clamp loader proteins

真核生物DNA复制过程中的DNA延伸反应需要一组复制蛋白，PCNA和RFC，它们分别起钳夹和装载蛋白的作用。到目前为止，已鉴定出它们的相关蛋白，如Rad 9 -1-1、rad 17、Chl 12、Mgs 1等，并有望作为一种新的钳位和装载蛋白发挥功能。由于它们与PCNA和RFC的相似性，它们将作为起源于DNA复制的各种DNA代谢途径的连接器发挥核心作用。为了阐明在DNA复制过程中维持基因组稳定的多个钳蛋白和钳装载蛋白网络的整体特征，我们研究了它们的结构和功能。首先，我们重构了一种新的钳蛋白复合物（9-1-1）和一种钳装载蛋白复合物（Rad 17-RFC），这两种蛋白都是人类检查点途径所必需的。我们进一步证明了它们分别与PCNA和RFC具有不可区分的结构。因此，特定的钳蛋白和装载蛋白将在检查点应答途径中具有特定的作用。第二，我们已经重建了一个装载机复杂的染色体凝聚因子，Chl 12，并证明了这个复杂的功能作为第二个PCNA装载机。第三，我们纯化了一个RFC相关蛋白，WRNIP 1，它已被确定为一个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。此外，由于它与DNA聚合酶δ特异性相互作用并刺激其活性，因此可能参与DNA复制过程中DNA合成的调控，这些结果强烈提示多种PCNA和RFC相关蛋白可能组成一个巨大的蛋白质网络来协调DNA复制，并通过多种途径实现精确的DNA复制和染色体分离。

DNA elongation reaction during eukaryotic DNA replication requires a set of replication proteins, PCNA and RFC, which function as a clamp and its loader protein, respectively. So far, their related proteins have been identified, for example, Rad9-1-1,rad17,Chl12,Mgs1, and expected to function as a novel clamp and loader proteins. Due to their similarities with PCNA and RFC, they will have central roles as connectors of various DNA metabolic pathways originated from DNA replication. To elucidate a whole feature of this network of multiple clamp and clamp loader proteins to maintain genome stability during DNA replication, we have studied on their structures and functions.First, we have reconstituted a novel clamp complex (9-1-1) and a loader complex (Rad17-RFC), both of which are required for the human checkpoint pathway. We further demonstrated that they have indistinguishable structures from those of PCNA and RFC, respectively. Thus, a specific clamp and loader proteins will have a particular role in the checkpoint response pathway. Second, we have reconstituted a loader complex with chromosome cohesion factor, Chl12, and demonstrated that this complex functions as the second PCNA loader. Third, we 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. Furthermore, since it interacts specifically with DNA polymerase δ and stimulates the activity, it will be involved in regulation of DNA synthesis during DNA replication.These results strongly suggest that multiple PCNA- and RFC-related proteins would organize a huge protein network to coordinate DNA replication and various pathways for precise DNA replication and chromosome segregation.