Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases
Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases
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DOI：
10.1093/nar/gkw1130
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发表时间：
2017-01-01
影响因子：
14.9
通讯作者：
Glickman, Michael S.
中科院分区：
文献类型：
--
作者：
Gupta, Richa;Unciuleac, Mihaela-Carmen;Glickman, Michael S.
目前的细菌同源重组(HR)模型认为，通过多亚单位解旋酶-核酸酶机器(如RecBCD、AddAB或AdnAB)广泛切除DNA双链断裂(DSB)，可以产生RecA介导的链侵袭所需的3‘单链DNA底物。AdnAB是与分枝杆菌HR相关的解旋酶-核酸酶，由aDNA和AdnB两个亚基组成，每个亚基由一个N-端ATPase结构域和一个C-末端核酸酶结构域组成。AdnAB在体外对DSB的解离严格依赖于位于3‘ssDNA链上的’先导‘AdnB马达的ATPase活性，而不是推测的’滞后‘aDNA ATPase。在这里，我们从遗传学上质疑AdnAB的哪些活性与其在体内HR和DNA损伤修复中的作用有关，通过灭活这四个催化域中的每一个。完全核酸酶死亡的AdnAB酶可以维持体内重组，只要它的AdnB马达完整，并且有Reco和RecR可用。我们得出结论：AdnAB在HR中的DSB解离活性足以满足AdnAB的功能。虽然不排除备用核酸酶的作用，但我们的发现表明，分枝杆菌HR可以通过DSB解离和蛋白质捕获移位的3‘-OH单链进行，而不需要广泛的末端切除。
Current models of bacterial homologous recombination (HR) posit that extensive resection of a DNA double-strand break (DSB) by a multisubunit helicase-nuclease machine (e.g. RecBCD, AddAB or AdnAB) generates the requisite 3' single-strand DNA substrate for RecA-mediated strand invasion. AdnAB, the helicase-nuclease implicated in mycobacterial HR, consists of two subunits, AdnA and AdnB, each composed of an N-terminal ATPase domain and a C-terminal nuclease domain. DSB unwinding by AdnAB in vitro is stringently dependent on the ATPase activity of the `lead' AdnB motor translocating on the 3' ssDNA strand, but not on the putative `lagging' AdnA ATPase. Here, we queried genetically which activities of AdnAB are pertinent to its role in HR and DNA damage repair in vivo by inactivating each of the four catalytic domains. Complete nuclease-dead AdnAB enzyme can sustain recombination in vivo, as long as its AdnB motor is intact and RecO and RecR are available. We conclude that AdnAB's processive DSB unwinding activity suffices for AdnAB function in HR. Albeit not excluding the agency of a backup nuclease, our findings suggest that mycobacterial HR can proceed via DSB unwinding and protein capture of the displaced 3' -OH single strand, without a need for extensive end-resection.