Wilkinson Martin, Troman Luca, Wan Nur Ismah Wan Ak, Chaban Yuriy, Avison Matthew B, Dillingham Mark S, Wigley Dale B
Department of Medicine, Section of Structural Biology, Imperial College London, London, United Kingdom.
School of Biochemistry, University of Bristol, Bristol, United Kingdom.
Elife. 2016 Dec 23;5:e22963. doi: 10.7554/eLife.22963.
Our previous paper (Wilkinson , 2016) used high-resolution cryo-electron microscopy to solve the structure of the RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in or increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.
我们之前的论文（威尔金森，2016年）使用高分辨率冷冻电子显微镜解析了RecBCD复合物的结构，该复合物在双链DNA断裂修复和噬菌体DNA降解中均发挥作用。为了对抗后者的活性，噬菌体λ编码一种名为Gam的小蛋白抑制剂，它与RecBCD结合并使该复合物失活。在此，我们表明Gam通过在DNA结合位点竞争来抑制RecBCD。相互作用表面广泛，涉及对DNA底物的分子模拟。我们还表明，在大肠杆菌或鼠伤寒沙门氏菌中表达Gam会增加对氟喹诺酮类药物的敏感性；氟喹诺酮类药物是一类通过抑制拓扑异构酶并诱导双链DNA断裂来杀死细胞的抗菌药物。此外，临床分离株中的氟喹诺酮耐药性可通过表达Gam来逆转。总之，我们的数据解释了拓扑异构酶诱导的DNA断裂与RecBCD基因产物之间观察到的合成致死性，提示了一种新的联合抗菌策略。
