Wang Jindong, Lindner Scott E, Leight Elizabeth R, Sugden Bill
McArdle Laboratory for Cancer Research, University of Wisconsin--Madison, Madison, WI 53706, USA.
Mol Cell Biol. 2006 Feb;26(3):1124-34. doi: 10.1128/MCB.26.3.1124-1134.2006.
We developed a mammalian plasmid replicon with a formerly uncharacterized origin of DNA synthesis, 8xRep*. 8xRep* functions efficiently to support once-per-cell-cycle synthesis of plasmid DNA which initiates within Rep*. By characterizing Rep*'s requirements for acting as an origin, we have uncovered several striking properties it shares with DS, the only other well-characterized, licensed, mammalian plasmid origin of DNA synthesis. Rep* contains a pair of previously unrecognized Epstein-Barr nuclear antigen 1 (EBNA1)-binding sites that are both necessary and sufficient in cis for its origin activity. These sites have an essential 21-bp center-to-center spacing, are bent by EBNA1, and recruit the origin recognition complex. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon. The role of EBNA1 likely reflects its evolution from cellular factors involved in the assembly of the initiation machinery.
我们开发了一种具有以前未表征的DNA合成起始位点8xRep的哺乳动物质粒复制子。8xRep能有效地发挥作用，以支持在Rep内起始的质粒DNA的单周期合成。通过表征Rep作为起始位点的条件，我们发现了它与DS（另一个唯一已充分表征、获得许可的哺乳动物质粒DNA合成起始位点）共有的几个显著特性。Rep包含一对以前未识别的爱泼斯坦-巴尔核抗原1（EBNA1）结合位点，这些位点对于其起始活性在顺式中既是必需的也是充分的。这些位点具有至关重要的21碱基对中心间距，被EBNA1弯曲，并招募起始识别复合物。DS和Rep之间共有的特性定义了哺乳动物染色体外复制子的顺式和反式特征。EBNA1的作用可能反映了它从参与起始机制组装的细胞因子进化而来。
