Chédin Frédéric, Handa Naofumi, Dillingham Mark S, Kowalczykowski Stephen C
Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616, USA.
J Biol Chem. 2006 Jul 7;281(27):18610-7. doi: 10.1074/jbc.M600882200. Epub 2006 Apr 21.
The Bacillus subtilis AddAB enzyme possesses ATP-dependent helicase and nuclease activities, which result in the unwinding and degradation of double-stranded DNA (dsDNA) upon translocation. Similar to its functional counterpart, the Escherichia coli RecBCD enzyme, it also recognizes and responds to a specific DNA sequence, referred to as Chi (chi). Recognition of chi triggers attenuation of the 3'- to 5'-nuclease, which permits the generation of recombinogenic 3'-overhanging, single-stranded DNA (ssDNA), terminating at chi. Although the RecBCD enzyme briefly pauses at chi, no specific binding of RecBCD to chi during translocation has been documented. Here, we show that the AddAB enzyme transiently binds to its cognate chi sequence (chi(Bs): 5'-AGCGG-3') during translocation. The binding of AddAB enzyme to the 3'-end of the chi(Bs)-specific ssDNA results in protection from degradation by exonuclease I. This protection is gradually reduced with time and lost upon phenol extraction, showing that the binding is non-covalent. Addition of AddAB enzyme to processed, chi(Bs)-specific ssDNA that had been stripped of all protein does not restore nuclease protection, indicating that AddAB enzyme binds to chi(Bs) with high affinity only during translocation. Finally, protection of chi(Bs)-specific ssDNA is still observed when translocation occurs in the presence of competitor chi(Bs)-carrying ssDNA, showing that binding occurs in cis. We suggest that this transient binding of AddAB to chi(Bs) is an integral part of the AddAB-chi(Bs) interaction and propose that this molecular event underlies a general mechanism for regulating the biochemical activities and biological functions of RecBCD-like enzymes.
枯草芽孢杆菌AddAB酶具有ATP依赖性解旋酶和核酸酶活性，在移位时会导致双链DNA（dsDNA）解旋和降解。与其功能对应物大肠杆菌RecBCD酶类似，它也能识别并响应一种特定的DNA序列，称为Chi（chi）。对chi的识别会触发3'至5'核酸酶活性的减弱，从而允许产生在chi处终止的具有重组活性的3'突出单链DNA（ssDNA）。尽管RecBCD酶在chi处会短暂停顿，但尚未有文献记载RecBCD在移位过程中与chi有特异性结合。在此，我们表明AddAB酶在移位过程中会短暂结合其同源chi序列（chi(Bs)：5'-AGCGG-3'）。AddAB酶与chi(Bs)特异性ssDNA的3'末端结合可保护其不被核酸外切酶I降解。这种保护会随着时间逐渐减弱，并在苯酚抽提后消失，表明这种结合是非共价的。将AddAB酶添加到已去除所有蛋白质的经过处理的chi(Bs)特异性ssDNA中并不能恢复核酸酶保护作用，这表明AddAB酶仅在移位过程中以高亲和力结合chi(Bs)。最后，当在携带竞争性chi(Bs)的ssDNA存在的情况下发生移位时，仍能观察到对chi(Bs)特异性ssDNA的保护作用，表明结合是在顺式中发生的。我们认为AddAB与chi(Bs)的这种短暂结合是AddAB-chi(Bs)相互作用的一个组成部分，并提出这一分子事件是调节RecBCD样酶生化活性和生物学功能的一般机制的基础。
