Loading mechanism of DNA polymerases to replication origin

芽殖酵母的Dpb11具有4个BRCT （BRCA1 C-Terminus）重复序列，其功能是蛋白质相互作用，是DNA复制和细胞周期检查点所必需的。我们还分离到dpb11-1的合成致死性Sld(synthetic lethality)1 ~ 6。在这项研究中，我们揭示了以下描述。1) Dpb11与DNA聚合酶ε形成复合体，是将DNA聚合酶装载到复制起点所必需的。2)当DNA复制被羟基脲抑制时，DNA复制酶与迟发起源的关联被阻断。在dpb11-1细胞中，像其他检查点突变体一样，该块被释放。因此，Dpb11似乎很可能通过阻滞迟发起源来为检查点起作用。3) Sld3与Cdc45一样，与G1期的早发起源和S期的晚发起源有关。4) Sld3和Cdc45形成一个复合体，这个复合体的形成是两种蛋白起源结合所必需的。5)起始点解绕需要Sld3和Cdc45的起始点关联。6) Sld5和Psf1 （Sld5的伴侣）在S期形成与复制起点相关的复合体，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，这种磷酸化依赖于S- cdk的活性。只有磷酸化形式的Sld2与Dpb11形成复合体，这是进一步装载DNA聚合酶所必需的。因此，S-Cdk通过磷酸化Sld2来控制DNA复制的起始。综上所述，Dpb11与Sld蛋白一起起着将DNA聚合酶装载到复制起点的作用。进一步的分析将对DNA聚合酶的起源链有更清晰的认识。

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.