Structure and primase-mediated activation of a bacterial dodecameric replicative helicase.
Structure and primase-mediated activation of a bacterial dodecameric replicative helicase.
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DOI：
10.1093/nar/gkv792
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发表时间：
2015-09-30
影响因子：
14.9
通讯作者：
Terradot L
中科院分区：
文献类型：
--
作者：
Bazin A;Cherrier MV;Gutsche I;Timmins J;Terradot L
复制解旋酶是解开DNA以启动染色体复制的必需ATP酶。虽然细菌复制DnaB解旋酶是六聚体，但幽门螺杆菌DnaB（HpDnaB）被发现形成双六聚体，类似于一些古细菌和真核生物复制解旋酶。在这里，我们提出了一个结构和功能分析的HpDnaB蛋白在primosome形成。HpDnaB在6.7 nm分辨率下的晶体结构揭示了由两个六聚体组成的十二聚体组织，所述六聚体通过它们的N-末端环以堆叠扭曲模式组装。使用荧光各向异性，我们表明，HpDnaB十二聚体与单链DNA在ATP的存在下相互作用，但具有低的DNA解旋活性。多角度光散射和小角度X射线散射表明，与DnaG引物解旋酶结合结构域的相互作用将解旋酶十二聚体解离成单环引物体。对蛋白质和相关复合物的功能测定表明，这些单环的primosomes是ATP水解，DNA结合和解旋的解旋酶的最活跃形式。这些发现揭示了引发酶对HpDnaB的激活机制，这可能与其他细菌和可能利用十二聚体解旋酶进行DNA复制的其他生物有关。
Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.