复制叉激活是由 CMG 解旋酶中的单链 DNA 门控实现的。
Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase.
机构信息
Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY 10065, USA.
Laboratory of DNA Replication, The Rockefeller University, New York, NY 10065, USA.
出版信息
Cell. 2019 Jul 25;178(3):600-611.e16. doi: 10.1016/j.cell.2019.06.032.
The eukaryotic replicative helicase CMG is a closed ring around double-stranded (ds)DNA at origins yet must transition to single-stranded (ss)DNA for helicase action. CMG must also handle repair intermediates, such as reversed forks that lack ssDNA. Here, using correlative single-molecule fluorescence and force microscopy, we show that CMG harbors a ssDNA gate that enables transitions between ss and dsDNA. When coupled to DNA polymerase, CMG remains on ssDNA, but when uncoupled, CMG employs this gate to traverse forked junctions onto dsDNA. Surprisingly, CMG undergoes rapid diffusion on dsDNA and can transition back onto ssDNA to nucleate a functional replisome. The gate-distinct from that between Mcm2/5 used for origin loading-is intrinsic to CMG; however, Mcm10 promotes strand passage by enhancing the affinity of CMG to DNA. This gating process may explain the dsDNA-to-ssDNA transition of CMG at origins and help preserve CMG on dsDNA during fork repair.
真核复制解旋酶 CMG 以双链 DNA（dsDNA）为闭合环在起始点，但必须转变为单链 DNA（ssDNA）以发挥解旋酶的作用。CMG 还必须处理修复中间体，如缺乏 ssDNA 的反向叉。在这里，我们使用相关的单分子荧光和力显微镜，表明 CMG 具有 ssDNA 门，可实现 ss 和 dsDNA 之间的转变。当与 DNA 聚合酶偶联时，CMG 会留在 ssDNA 上，但当解偶联时，CMG 会利用该门在分叉连接处进入 dsDNA。令人惊讶的是，CMG 在 dsDNA 上会发生快速扩散，并可返回 ssDNA 以引发功能性复制体。该门不同于用于起始加载的 Mcm2/5 之间的门，是 CMG 固有的；然而，Mcm10 通过增强 CMG 与 DNA 的亲和力来促进链通过。这个门控过程可以解释 CMG 在起始点处的 dsDNA 到 ssDNA 的转变，并有助于在叉修复过程中保持 CMG 在 dsDNA 上。
Zotero 插件