Structural basis for DNA strand separation by a hexameric replicative helicase.
Structural basis for DNA strand separation by a hexameric replicative helicase.
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DOI：
10.1093/nar/gkv778
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发表时间：
2015-09-30
影响因子：
14.9
通讯作者：
Orlova EV
中科院分区：
文献类型：
--
作者：
Chaban Y;Stead JA;Ryzhenkova K;Whelan F;Lamber EP;Antson A;Sanders CM;Orlova EV
六聚体解旋酶是进行性DNA解旋机器，但它们在解旋过程中如何与复制叉接合尚不清楚。使用电子显微镜和单粒子分析，我们确定了完整的六聚体解旋酶E1从乳头瘤病毒和两个复合物的E1绑定到一个DNA复制叉末端标记的蛋白质标签的结构。通过链霉亲和素（dsDNA末端）和Fab（5′ ssDNA）标记DNA复制叉，我们确定了这些标记在解旋酶表面的位置，表明至少有10 bp的dsDNA通过侧通道进入E1解旋酶。在目前公认的dsDNA解旋的“空间排斥”模型中，当dsDNA链在蛋白质组装体外楔入时，活性3′ ssDNA链被拉过解旋酶马达结构域的中心隧道。我们的结构观察和核酸酶足迹分析表明：链分离发生在E1内解旋酶结构域上方的一个腔室中，5′被动ssDNA链通过与dsDNA进入点相反的单独隧道离开组装体。因此，我们的数据表明，替代目前的一般模型的DNA解旋的六聚体解旋酶。
Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.