Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots
Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots
复制标题
DOI：
10.1101/gad.1605807
复制
发表时间：
2007-12-15
影响因子：
10.5
通讯作者：
Smith, Gerald R.
中科院分区：
文献类型：
--
作者：
Amundsen, Susan K.;Taylor, Andrew F.;Smith, Gerald R.
大肠杆菌RecBCD解旋酶-核酸酶是复杂蛋白机器的范例，启动同源基因重组和修复断裂的DNA。从双链体末端开始，RecBCD在互补链上用其快速RecD解旋酶和较慢的RecB解旋酶解旋DNA。当遇到Chi热点（5 '-GCTGGTGG-3'）时，该酶产生一个新的3'单链末端并将RecA蛋白加载到其上，但Chi如何调节RecBCD尚不清楚。我们报告了一类新的突变体RecBCD酶，其在依赖于DNA底物长度的新位置切割DNA，并且与RecB：RecD解旋酶速率严格相关。我们的结论是，在突变酶中，当RecD到达DNA末端时，它向RecB的核酸酶结构域发出信号以切割DNA。正如这种解释所预测的那样，当解旋被另一个反向运动的RecBCD分子阻断时，突变酶会更靠近DNA上的进入点切割。此外，当RecD被改变其ATP酶位点的突变减慢时，使得RecB在RecD之前到达DNA末端，长度依赖性切割被消除。这些观察结果使我们假设，在野生型RecBCD酶中，Chi被RecC识别，然后RecC发出信号让RecD停止，RecD又发出信号让RecB切割DNA并装载RecA。我们讨论支持这种“信号级联”的假设和测试。亚基间信号可能会调节其他复杂的蛋白质机器。
The Escherichia coli RecBCD helicase-nuclease, a paradigm of complex protein machines, initiates homologous genetic recombination and the repair of broken DNA. Starting at a duplex end, RecBCD unwinds DNA with its fast RecD helicase and slower RecB helicase on complementary strands. Upon encountering a Chi hot spot (5'-GCTGGTGG-3'), the enzyme produces a new 3' single-strand end and loads RecA protein onto it, but how Chi regulates RecBCD is unknown. We report a new class of mutant RecBCD enzymes that cut DNA at novel positions that depend on the DNA substrate length and that are strictly correlated with the RecB: RecD helicase rates. We conclude that in the mutant enzymes when RecD reaches the DNA end, it signals RecB's nuclease domain to cut the DNA. As predicted by this interpretation, the mutant enzymes cut closer to the entry point on DNA when unwinding is blocked by another RecBCD molecule traveling in the opposite direction. Furthermore, when RecD is slowed by a mutation altering its ATPase site such that RecB reaches the DNA end before RecD does, the length-dependent cuts are abolished. These observations lead us to hypothesize that, in wild-type RecBCD enzyme, Chi is recognized by RecC, which then signals RecD to stop, which in turn signals RecB to cut the DNA and load RecA. We discuss support for this "signal cascade" hypothesis and tests of it. Intersubunit signaling may regulate other complex protein machines.