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Regulatory Mechanism of Activation of MCM Helicase in DNA Replication Initiation

DNA复制起始中MCM解旋酶激活的调控机制

基本信息

批准号：
17570124
负责人：
YOU Zhiying
金额：
$ 2.24万
依托单位：
Tokyo Metropolitan Organization for Medical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570124/
关键词：
DNA Replication MCM Helicase Cdt1 Protein Replication Fork MCM Binding-protein DNA-binding Activity Cdt1

项目摘要

MCM (minichromosome maintenance) plays central roles in initiation and elongation of DNA replication as a replicative helicase. We previously reported that the helicase activity of mouse Mcm4/6/7 complex is activated specifically by thyimine-rich single-stranded DNA. In this research, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5'-proximal 25 nucleotide single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds to various fork and extension structures with similar affinity as long as they contain a single-stranded DNA segment. Strands are displaced, however, only when it is loaded … More onto a single-stranded 3'-tail, permitting its translocation from 3' to 5'. The extent of helicase activation depends on the sequence context of the 3'-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine-guanine pairs with cytosine-inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex (You ＆ Masai, NAR 2005).On the other hand, the Mcm helicase is expected to interact with many factors during the loading and translocation on the template DNA. Cdt1 is one of them. We found that the Mcm complex including Mcm2-7, Mcm4/6/7 and Mcm2/4/6/7 cosediment with the Cdt1 in the glycerol gradient centrifugation. Cdt1 interacts with a single polypeptide of Mcm2 as well as with the Mcm4/6 complex. Cdt1 is an ATP binding protein and forms a big complex with Mcm in the presence of ATP. The DNA binding and helicase activities of Mcm4/6/7 were dramatically stimulated by Cdt1 protein in vitro, suggesting Cdt1 facilitates the loading of Mcm onto chromatin. These facts suggest that Cdt1 has a similar function to DnaC that is the loading factor of E.coli DnaB helicase. Less

MCM（微型染色体维持）作为复制解旋酶在 DNA 复制的起始和延长中发挥着核心作用。我们之前报道过，小鼠Mcm4/6/7复合物的解旋酶活性是由富含胸腺嘧啶的单链DNA特异性激活的。在这项研究中，我们使用各种合成 DNA 检查了其底物偏好和解旋酶作用。在气泡底物上，Mcm4/6/7 与分支点附近的 5'-近端 25 个核苷酸单链片段进行对称双重接触，大概生成双六聚体。一条单链胸腺嘧啶残基的丢失导致解旋效率显着降低，这表明单个双六聚体 Mcm4/6/7 的同时双向解旋可能在气泡的有效解旋中发挥作用。 Mcm4/6/7 能够以相似的亲和力与各种叉和延伸结构结合，只要它们包含单链 DNA 片段。然而，只有当它被加载到单链 3' 尾上时，链才会发生移位，从而允许其从 3' 易位到 5'。解旋酶激活的程度取决于 3'-尾的序列背景，胸腺嘧啶含量为 50% 或更多的 DNA 可以达到最大水平。随着双链体区域 GC 含量的增加，Mcm4/6/7 的链置换受到抑制。双链体中胞嘧啶-鸟嘌呤对替换为胞嘧啶-肌苷对可恢复解旋，表明哺乳动物 Mcm4/6/7 解旋酶难以解开稳定的碱基配对双链体 (You ＆ Masai, NAR 2005)。另一方面，Mcm 解旋酶预计在模板 DNA 上的加载和易位过程中与许多因素相互作用。 Cdt1 就是其中之一。甘油梯度离心发现Mcm复合物包括Mcm2-7、Mcm4/6/7和Mcm2/4/6/7与Cdt1共沉淀。 Cdt1 与 Mcm2 的单个多肽以及 Mcm4/6 复合物相互作用。 Cdt1 是一种 ATP 结合蛋白，在 ATP 存在的情况下与 Mcm 形成大复合物。在体外，Cdt1 蛋白显着刺激 Mcm4/6/7 的 DNA 结合和解旋酶活性，表明 Cdt1 有助于将 Mcm 装载到染色质上。这些事实表明，Cdt1 具有与 DnaC 类似的功能，即大肠杆菌 DnaB 解旋酶的装载因子。较少的