Loading mechanism of DNA polymerases to replication origin

DNA聚合酶复制起点的加载机制

基本信息

批准号：
11694331
负责人：
ARAKI Hiroyuki
金额：
$ 5.25万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11694331/
关键词：
DNA Replication Cell Cycle DNA Polymerase BRCT BRCT 複製開始チエックポイント

项目摘要

Dpb11 of budding yeast has four BRCT (BRCA1 C-Terminus) repeats, which function for protein-protein interactions, and it is required for DNA replication and cell cycle checkpoint. We also isolated Sld(synthetic lethality with dpb11-1)1〜6. In this study, we revealed those described below.1) Dpb11 forms a complex with DNA polymerase ε and is required for loading of DNA polymerase to replication origins. 2) When DNA replication is inhibited by hydroxyl urea, associations of DNA replication enzymes with late-firing origins are blocked. In dpb11-1 cells, this block is released, like other checkpoint mutants. Thus, it seems likely that Dpb11 functions for the checkpoint through the block of late-firing origins. 3) Sld3, like Cdc45, associates with early-firing origins in G1 phase and with late-firing origins in S phase. 4) Sld3 and Cdc45 form a complex and this complex formation is required for origin associations of both proteins. 5) The origin associations of Sld3 and Cdc45 is required for the origin unwinding. 6) Sld5 and Psf1 (Partner of sld five) form a complex which associates with replication origins in S phase and is required for origin association of DNA polymerase.7) Sld2 which form a complex with Dpb11 is phosphorylated in S phase and this phosphorylation depends on S-Cdk activity. Only the phosphorylated form of Sld2 form a complex with Dpb11, which is required for further loading of DNA polymerase. Thus, S-Cdk controls the initiation of DNA replication through phosphorylation of Sld2. Taken all together, Dpb11 together with Sld proteins functions for loading of DNA polymerase to replication origins. Future analysis will give a clear view for origin lading of DNA polymerase.

芽殖酵母的 Dpb11 有 4 个 BRCT (BRCA1 C-Terminus) 重复序列，其功能是蛋白质-蛋白质相互作用，是 DNA 复制和细胞周期检查点所必需的。我们还分离出了 Sld(synthetic lethality with dpb11-1)1～6。在这项研究中，我们揭示了以下内容。1) Dpb11 与 DNA 聚合酶 ε 形成复合物，并且是将 DNA 聚合酶加载到复制起点所必需的2)。当 DNA 复制被羟基脲抑制时，DNA 复制酶与晚激发起点的关联被阻断，在 dpb11-1 细胞中，该阻断被释放，就像其他检查点突变体一样，因此，Dpb11 似乎通过检查点发挥作用。 3) Sld3 与 Cdc45 一样，与 G1 期的早发起源相关，并与 S 期的晚发起源相关。 Sld3 和 Cdc45 形成复合物，并且这种复合物的形成是两种蛋白质的起源关联所必需的。 5) Sld3 和 Cdc45 的起源关联是起源解旋所必需的。 6) Sld5 和 Psf1（sld 5 的伙伴）形成复合物。与 S 期的复制起点相关，并且是 DNA 聚合酶起点关联所必需的。 7) 与 Dpb11 形成复合物的 Sld2 在 S 期被磷酸化，这磷酸化取决于 S-Cdk 活性。只有 Sld2 的磷酸化形式才能与 Dpb11 形成复合物，这是进一步加载 DNA 聚合酶所需的。因此，S-Cdk 通过 Sld2 的磷酸化控制 DNA 复制的启动。 Dpb11 与 Sld 蛋白一起发挥 DNA 聚合酶装载到复制起点的作用，未来的分析将为 DNA 聚合酶的装载提供清晰的视角。