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.

发芽酵母的DPB11具有四个BRCT（BRCA1 C-末端）重复序列，这在蛋白质 - 蛋白质相互作用中起作用，并且是DNA复制和细胞周期检查点所必需的。我们还分离了SLD（与DPB11-1的合成致死性）1-6。在这项研究中，我们揭示了下面描述的。1）DPB11形成了具有DNA聚合酶ε的复合物，是将DNA聚合酶加载至复制起源所必需的。 2）当羟基尿素抑制DNA复制时，DNA复制酶与晚期起源的关联被阻断。在DPB11-1细胞中，与其他检查点突变体一样，该块被释放。这是DPB11通过晚期起源的块为检查点起作用的。 3）SLD3，例如CDC45，将与G1相结合，并在S相延迟起源。 4）SLD3和CDC45形成一个复杂的形成，并且这种复杂的形成是两种蛋白质的起源关联所必需的。 5）SLD3和CDC45的原点关联是原点解放所必需的。 6）SLD5和PSF1（SLD 5的合作伙伴）形成了与复制起源相关的复合体，并且是DNA聚合酶的起源相关性所必需的。7）SLD2与DPB11形成复合物在S相中磷酸化，并且该磷酸化取决于S-CDK活性。仅SLD2的磷酸化形式与DPB11形成复合物，这是进一步加载DNA聚合酶所必需的。这是S-CDK通过SLD2的磷酸化来控制DNA复制的启动。全部将DPB11与SLD蛋白功能一起使用，用于将DNA聚合酶载入复制起源。未来的分析将对DNA聚合酶起源的起源有清晰的看法。