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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Dpb 11含有4个BRCT（BRCA 1 C-Terminus）重复序列，其功能是蛋白质间相互作用，是DNA复制和细胞周期检查点所必需的。我们还分离出Sld（dpb 11 -1的合成致死率）1 × 10 - 6。1）Dpb 11与DNA聚合酶ε形成复合物，是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复制酶与迟发起点的关联被阻断。在dpb 11 -1细胞中，这种阻断被释放，就像其他检查点突变体一样。因此，Dpb 11似乎很可能通过延迟激发起源的阻断来发挥检查点的作用。3)Sld 3与Cdc 45一样，在G1期与早放电起源相关，在S期与晚放电起源相关。4)Sld 3和Cdc 45形成复合物，这种复合物的形成是两种蛋白质的起源关联所必需的。5)Sld 3和Cdc 45的起源关联是起源解旋所必需的。6)Sld 5和Psf 1（Sld 5的配偶体）在S期与复制起点结合，是DNA聚合酶的起点结合所必需的。7）Sld 2与Dpb 11结合，在S期被磷酸化，这种磷酸化依赖于S-Cdk活性。只有Sld 2的磷酸化形式与Dpb 11形成复合物，这是进一步装载DNA聚合酶所必需的。因此，S-Cdk通过Sld 2的磷酸化来控制DNA复制的起始。总之，Dpb 11与Sld蛋白一起用于将DNA聚合酶装载到复制起点。未来的分析将为DNA聚合酶的起源定位提供一个清晰的视角。