Regulatory Mechanism of Activation of MCM Helicase in DNA Replication Initiation

微小染色体维持(MCM)作为一种复制型解旋酶，在DNA复制的起始和延长过程中起着核心作用。我们以前报道过小鼠Mcm4/6/7复合体的解旋酶活性被富含胸腺亚胺的单链DNA特异性激活。在这项研究中，我们使用不同的合成DNA来研究它的底物偏好和解旋酶的作用。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的双重接触，可能产生双六角体。单链上胸腺嘧啶残基的丢失会导致解离效率的显著降低，这表明由单个双六聚体Mcm4/6/7同时进行的双向解离可能在有效解离气泡方面发挥作用。Mcm4/6/7以相似的亲和力结合到各种分叉和延伸结构上，只要它们包含单链DNA片段。但是，只有在加载了…时，链才会被置换更多的是单链的3‘-尾巴，允许它从3’到5‘的移位。解旋酶的激活程度取决于3‘-尾的序列背景，最大水平是胸腺嘧啶含量为50%或更多的DNA。随着双链区GC含量的增加，Mcm4/6/7的链置换受到抑制。在双链中用胞嘧啶-鸟苷对取代胞嘧啶-鸟氨酸对恢复解离，这表明哺乳动物Mcm4/6/7解旋酶在稳定地解开碱基配对的双链时存在困难(You&Masai，NAR 2005)。另一方面，MCM解旋酶在模板DNA的装载和转位过程中预计会与许多因素相互作用。CDT1就是其中之一。在甘油梯度离心法中，我们发现包括Mcm2-7、Mcm4/6/7和Mcm2/4/6/7在内的MCM复合体与CDt1共表达。CDT1不仅与Mcm4/6复合体相互作用，而且与McM2的单一多肽相互作用。CDT1是一种三磷酸腺苷结合蛋白，在三磷酸腺苷存在下与MCM形成一个大的复合体。体外培养的Mcm4/6/7细胞的DNA结合活性和解旋酶活性均被CDT1蛋白显著刺激，提示CDT1促进了MCM在染色质上的负载。这些事实表明，CDT1具有与DNAC相似的功能，即大肠杆菌DNAB解旋酶的负载因子。较少

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