Regulatory Mechanism of Activation of MCM Helicase in DNA Replication Initiation

MCM（小染色体维持）作为一种复制解旋酶，在DNA复制的起始和延伸过程中起着核心作用。我们以前报道过小鼠Mcm4/6/7复合体解旋酶活性被胸腺嘧啶丰富的单链DNA特异性激活。在本研究中，我们使用各种合成dna检测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25个核苷酸单链片段进行对称的双接触，可能产生双六聚体。单链胸腺嘧啶残基的丢失导致解绕效果显著降低，这表明单个双六聚体Mcm4/6/7同时双向解绕可能在有效解绕气泡中起作用。Mcm4/6/7只要包含单链DNA片段，就能以相似的亲和力与各种分叉和延伸结构结合。然而，只有当它被加载到单链3‘尾部时，它才会发生位移，从而允许它从3’转移到5'。解旋酶的激活程度取决于3'尾部的序列背景，胸腺嘧啶含量达到50%或以上的DNA达到最大水平。随着双相区GC含量的增加，Mcm4/6/7的链位移受到抑制。用胞嘧啶-肌苷对取代双链中的胞嘧啶-鸟嘌呤对可以恢复解绕，这表明哺乳动物Mcm4/6/7解旋酶难以稳定地解绕碱基配对的双链（You & Masai, NAR 2005）。另一方面，Mcm解旋酶预计在模板DNA的装载和易位过程中与许多因素相互作用。Cdt1就是其中之一。在甘油梯度离心中，我们发现Mcm复合物包括Mcm2-7、mcm2 /6/7和Mcm2/4/6/7与Cdt1共沉。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存在下与Mcm形成大复合体。Cdt1蛋白显著刺激Mcm4/6/7的DNA结合和解旋酶活性，表明Cdt1促进了Mcm在染色质上的加载。这些事实表明，Cdt1与大肠杆菌dna解旋酶的装载因子DnaC具有相似的功能。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