Synergy between RecBCD subunits is essential for efficient DNA unwinding

The subunits of the bacterial RecBCD act in coordination, rapidly and processively unwinding DNA at the site of a double strand break. RecBCD is able to displace DNA binding proteins, suggesting that it generates high forces, but the specific role of each subunit in the force generation is unclear. Here, we present a novel optical tweezers assay that allows monitoring the activity of RecBCD's individual subunits, when they are part of an intact full complex. We show that RecBCD and its subunits are able to generate forces up to 25-40 pN without a significant effect on their velocity. Moreover, the isolated RecD translocates fast, but is a weak helicase with limited processivity. Experiments at a broad range of [ATP] and forces suggest that RecD unwinds DNA as a Brownian ratchet, rectified by ATP binding, and that the presence of the other subunits shifts the ratchet equilibrium towards the post-translocation state.

细菌RecBCD复合物（RecBCD）的各个亚基协同发挥作用，可在双链断裂位点快速且持续地解旋DNA。RecBCD能够置换DNA结合蛋白，这提示其可产生较高的作用力，但各亚基在力产生过程中的具体功能尚不明确。本研究开发了一种新型光镊检测方法，可在完整复合物状态下监测RecBCD单个亚基的活性。实验结果表明，RecBCD及其亚基可产生高达25~40皮牛（pN）的作用力，且该作用力对其运动速率无显著影响。此外，单独分离的RecD亚基移动速度较快，但属于解旋能力较弱且持续合成能力有限的解旋酶（helicase）。在宽泛的三磷酸腺苷（ATP）浓度与作用力范围内开展的实验显示，RecD通过布朗棘轮（Brownian ratchet）机制解旋DNA，该过程由ATP结合进行整流；而其他亚基的存在可将棘轮平衡向易位后状态偏移。