Department of Genetic Resources Technology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581, Japan.
Extremophiles. 2010 Jan;14(1):21-31. doi: 10.1007/s00792-009-0284-9. Epub 2009 Sep 30.
The initiation step is a key process to regulate the frequency of DNA replication. Although recent studies in Archaea defined the origin of DNA replication (oriC) and the Cdc6/Orc1 homolog as an origin recognition protein, the location and mechanism of duplex opening have remained unclear. We have found that Cdc6/Orc1 binds to oriC and unwinds duplex DNA in the hyperthermophilic archaeon Pyrococcus furiosus, by means of a P1 endonuclease assay. A primer extension analysis further revealed that this localized unwinding occurs in the oriC region at a specific site, which is 12-bp long and rich in adenine and thymine. This site is different from the predicted duplex unwinding element (DUE) that we reported previously. We also discovered that Cdc6/Orc1 induces topological changes in supercoiled oriC DNA, and that this process is dependent on the AAA+ domain. These results indicate that topological alterations of oriC DNA by Cdc6/Orc1 introduce a single-stranded region at the 12-mer site, that could possibly serve as an entry point for Mcm helicase.
起始步骤是调节 DNA 复制频率的关键过程。尽管最近在古菌中对 DNA 复制起始点（oriC）和 Cdc6/Orc1 同源物作为起始识别蛋白进行了研究，但双链打开的位置和机制仍不清楚。我们发现，通过 P1 内切酶测定，Cdc6/Orc1 在嗜热古菌 Pyrococcus furiosus 中与 oriC 结合并解开双链 DNA。引物延伸分析进一步表明，这种局部解旋发生在 oriC 区域的特定位置，该位置长 12 个碱基，富含腺嘌呤和胸腺嘧啶。该位点与我们之前报道的预测双链解开元件（DUE）不同。我们还发现 Cdc6/Orc1 诱导超螺旋 oriC DNA 的拓扑变化，并且该过程依赖于 AAA+结构域。这些结果表明，Cdc6/Orc1 对 oriC DNA 的拓扑改变在 12 -mer 位点引入了单链区域，该区域可能作为 Mcm 解旋酶的进入点。
