Itou Hiroshi, Yagura Masaru, Shirakihara Yasuo, Itoh Tateo
From the Structural Biology Center, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan,
the Department of Cell Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan, and.
J Biol Chem. 2015 Feb 6;290(6):3601-11. doi: 10.1074/jbc.M114.595645. Epub 2014 Dec 23.
Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.
双链DNA在复制前通常会被蛋白质寡聚体解开。质粒ColE2 - P9的Rep蛋白（34 kDa）是质粒DNA复制的必需起始因子。该蛋白作为单体以序列特异性方式结合复制起点（Ori）并解开DNA。在此，我们展示了与Ori DNA结合的Rep的DNA结合结构域（E2Rep - DBD）的晶体结构。该结构揭示了E2Rep - DBD对Ori特异性识别的基础，还表明它通过其三个连续结构模块的协同作用解开DNA。该结构还表明，形成紧密模块的功能未知的PriCT结构域在DNA解旋中起核心作用。一些古真核引发酶C末端PriCT结构域的保守性表明它可能在这些蛋白质中发挥类似作用。因此，这是第一份为保守的PriCT结构域的功能重要性提供结构基础的报告，同时也揭示了单一蛋白质解开DNA的新机制。
