Structural basis for translocation by AddAB helicase-nuclease and its arrest at χ sites

摘要

在细菌双螺旋DNA断裂修复中，自由端最初由一种双功能的螺旋酶/核酸酶作用于其上，典型情况就是由大肠杆菌的RecBCD酶作用于其上。由于RecBCD会将DNA展开，所以它最终将碰上一个名为Chi(χ)的极性八聚序列，后者会造成核酸酶活性减弱，使其特异性发生变化。现在，Dale Wigley及同事获得了来自枯草杆菌、与含χ的DNA相结合的一种相关的酶AddAB的几个结构。这些结构有助于认识转位过程、χ的识别以及当χ被识别时所出现的暂停。%In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence χ (Chi) and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease (reviewed in refs 3, 4). These enzyme complexes unwind and digest the DNA duplex from the broken end until they encounter a χ sequence, whereupon they produce a 3' single-stranded DNA tail onto which they initiate loading of the RecA protein. Consequently, regulation of the AddAB/ RecBCD complex by χ is a key control point in DNA repair and other processes involving genetic recombination. Here we report crystal structures of Bacillus subtilis AddAB in complex with different χ-containing DNA substrates either with or without a non-hydrolysable ATP analogue. Comparison of these structures suggests a mechanism for DNA translocation and unwinding, suggests how the enzyme binds specifically to χ sequences, and explains how χ recognition leads to the arrest of AddAB (and RecBCD) translocation that is observed in single-molecule experiments.