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

通过多个钳和钳加载蛋白研究 DNA 复制和 DNA 损伤反应之间的界面

基本信息

批准号：
14380328
负责人：
TSURIMOTO Toshiki
金额：
$ 9.28万
依托单位：
KYUSHU UNIVERSITY (2003-2004)Nara Institute of Science and Technology (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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.

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，它们分别起夹钳和装载蛋白的作用。目前，已经鉴定出了它们的相关蛋白，如Rad9-1-1、rad17、Chl12、Mgs1，并有望作为一种新型的夹紧和装载蛋白。由于它们与PCNA和RFC的相似性，它们将作为源自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性的多重箝位和箝位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，我们重组了一种新的夹紧复合物（9-1-1）和装载复合物（Rad17-RFC），这两种复合物都是人类检查点通路所必需的。我们进一步证明它们分别与PCNA和RFC具有难以区分的结构。因此，特定的夹紧和装载蛋白将在检查点反应途径中发挥特殊作用。其次，我们重组了一个带有染色体内聚因子Chl12的装载复合体，并证明了该复合体作为第二个PCNA装载器的功能。第三，我们纯化了一个rfc相关蛋白WRNIP1，该蛋白已被鉴定为Werner DNA解旋酶结合蛋白。该蛋白形成自寡聚复合物，并表现出atp酶活性。此外，由于它特异性地与DNA聚合酶δ相互作用并刺激活性，它将参与DNA复制过程中DNA合成的调节。这些结果强烈提示，多个PCNA和rfc相关蛋白将组织一个巨大的蛋白质网络来协调DNA复制和各种途径，以实现精确的DNA复制和染色体分离。

项目成果

期刊论文数量（37）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A proteomics approach to identify PCNA binding proteins in human cell lysates : identification of the human CHL12/RFCs2-5 complex as a novel PCNA binding protein.

鉴定人细胞裂解物中 PCNA 结合蛋白的蛋白质组学方法：将人 CHL12/RFCs2-5 复合物鉴定为新型 PCNA 结合蛋白。

DOI：
发表时间：
2002
期刊：
J.Biol.Chem. 277
影响因子：
0
作者：
Ohta;S.;Shiomi;Y.;Sugimoto;K.;Obuse;C.;Tsurimoto;T.
通讯作者：
T.

The reconstituted human Chl12-RFC complex functions as a second PCNA loader

重组的人类 Chl12-RFC 复合物充当第二个 PCNA 装载机

DOI：
发表时间：
2004
期刊：
Genes Cells 9
影响因子：
0
作者：
H.Nishitani1;N.Sugimoto;V.Roukos;Y.Nakanishi;M.Saijo;C.Obuse;T.Tsurimoto;K.I.Nakayama;K.Nakayama;M.Fujita;Z.Lygerou;T.Nishimoto;Obuse C.;Shiomi Y.
通讯作者：
Shiomi Y.

Scheduled Conversion of Replication Complex Architecture at Replication Origins of S.cerevisiae during the cell cycle.

细胞周期期间酿酒酵母复制起点处复制复合体结构的预定转换。