Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection
Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection
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DOI：
10.1073/pnas.1913546116
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发表时间：
2019-12-03
影响因子：
11.1
通讯作者：
Shuman, Stewart
中科院分区：
文献类型：
--
作者：
Jia, Ning;Unciuleac, Mihaela C.;Shuman, Stewart
分枝杆菌AdnAB是一种异二聚体解旋酶-核酸酶，其通过切除DNA双链断裂（DSB）来启动同源重组。AdnA和AdnB亚基各自由N-末端马达结构域和C-末端核酸酶结构域组成。在这里，我们报告冷冻电子显微镜（cryo-EM）结构的AdnAB在三种功能状态：在DNA的情况下，并在复杂的分叉双链DNA的5'单链DNA（ssDNA）的尾部切割之前和之后的AdnA核酸酶。这些结构揭示了5' ssDNA通过AdnA核酸酶结构域的路径和5'链切割的机制; 3'跟踪链通过AdnB马达和将ATP水解与机械功偶联的DNA接触的路径; AdnA铁-硫簇亚结构域在Y连接处的位置及其在维持解绕的5'和3'链的分裂轨迹中的可能作用。DSB切除的单分子DNA帘幕分析显示，AdnAB是高度进行性的，但倾向于在双链体DNA上的随机位点自发暂停。AdnAB的一个显著特性是DSB切除的速度在酶经历自发暂停后减慢。我们的研究结果突出了AdnAB与细菌DSB切除运动核酸酶的RecBCD和AddAB分支的共同和独特特性。
Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-a-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.