Molecular mechanism of assembly of replication proteins at replication origins in eukaryotes

真核生物复制起点组装复制蛋白的分子机制

• 批准号: 15207012
• 负责人: ARAKI Hiroyuki
• 金额: $ 31.53万
• 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: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15207012/
• 关键词: DNA replication; Initition of DNA replication; Complex formation; Yeast; CDK; 複製; 複製タンパク質; 染色体DNA; リン酸化; 複合体

At replication origins in eukaryotic cells, the pre-Replicative Complex (pre-RC) forms from late M phase to G1 phase when CDK activity is low. When CDK is activated at late G1 phase, many replication proteins assemble on the pre-RC to initiate DNA replication. However, how they assemble at origins had not been elucidated. This is because CDK substrates and CDK-dependent reaction at the initiation step of DNA replication was not known. In this study, we first identified Sld2 and Sld3 as CDK substrates essential for initiation of DNA replication. Both CDK-phosphorylated Sld2 and Sld3 bind to Dpbll. These bindings are essential for initiation of DNA replication, so that when both binding are bypassed DNA replication initiates in the absence of CDK activity. Sld2 has 11 CDK phosphorylation motifs. Phosphorylation of Thr84 at one of these motifs is a single determinant for binding to Dpbll while other Sld2 phosphorylations are prerequisites for Thr84 phosphorylation. This regulation seems to contribute to fine-tuning of origin firing. Sld3 forms a complex with a novel factor, Sld7 and functions for initiation of DNA replication. Next, we found formation of the pre-Loading Complex (pre-LC) as a CDK-dependent reaction. The pre-LC is a novel complex containing DNA polymerase ε, GINS, Sld2 and Dpbll, which formation depends on CDK activity but not association with origins or the pre-RC. We thus propose the model; when Sld2 is phosphorylated by CDK and binds to Dpbll the pre-LC forms; since Sld3 associates with origins in G1 phase it is phosphorylated by CDK at origins; the phosphorylated Sld3 recruits the pre-LC to origins through Dpbll; then, DNA replication initiates.

在真核细胞的复制起点，在CDK活性较低的M期晚期至G1期形成pre- replication Complex （pre-RC）。当CDK在G1期后期被激活时，许多复制蛋白聚集在pre-RC上启动DNA复制。然而，它们在起源上是如何聚集的还没有被阐明。这是因为DNA复制起始阶段的CDK底物和CDK依赖反应尚不清楚。在这项研究中，我们首先确定了Sld2和Sld3是DNA复制起始所必需的CDK底物。cdk磷酸化的Sld2和Sld3都能与Dpbll结合。这些结合对于DNA复制的启动至关重要，因此当两个结合都被绕过时，DNA复制就会在没有CDK活性的情况下启动。Sld2有11个CDK磷酸化基序。Thr84在其中一个基序上的磷酸化是与dpll结合的单一决定因素，而其他Sld2磷酸化是Thr84磷酸化的先决条件。这一规则似乎有助于原点点火的微调。Sld3与一种新的因子Sld7形成复合体，并起DNA复制起始的作用。接下来，我们发现预加载复合物（pre-LC）的形成是cdk依赖的反应。pre-LC是一种含有DNA聚合酶ε、GINS、Sld2和Dpbll的新型复合物，其形成依赖于CDK活性，而与起源或pre-RC无关。因此，我们提出这个模型；当Sld2被CDK磷酸化并与dpll结合时，前lc形成；由于Sld3在G1期与起源相关，它在起源处被CDK磷酸化；磷酸化的Sld3通过Dpbll将前lc招募到原点；然后，DNA复制开始。

项目成果

The role of CDK in the initiation step of DNA replication in eukaryotes.

• DOI: 10.1186/1747-1028-2-16
• 发表时间: 2007-06-05
• 期刊: Cell division
• 影响因子: 2.3
• 作者: Tanaka S;Tak YS;Araki H
• 通讯作者: Araki H

クロマチンと遺伝子機能制御

染色质和基因功能调控

• 发表时间: 2003
• 作者: 上村陽一郎;荒木弘之
• 通讯作者: 荒木弘之

DNA複製・修復がわかる

了解 DNA 复制和修复

• 发表时间: 2004
• 作者: 槌田 謙;小松賢志
• 通讯作者: 小松賢志

Cell Division

• DOI: 10.1038/164131a0
• 发表时间: 2021-06
• 期刊: Nature
• 影响因子: 64.8
• 作者: Rani Gupta;Namita Gupta;Amuliya Kashyap

ACDK-catalysed phosphorylation of the DNA replication complex Sld2-Dpbll

ACDK 催化 DNA 复制复合物 Sld2-Dpbll 的磷酸化

• 发表时间: 2006
• 期刊: The EMBO Journal Vol.25 No.9
• 作者: Price;P.W.;Ohgushi;T.;Roinine;H.;Ishihara;M.;Craig;T.P.;Tahvanainen;J.;Ferrier,S.M.;Yon-Soo Tak
• 通讯作者: Yon-Soo Tak