The AddAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation
The AddAB helicase/nuclease forms a stable complex with its cognate χ sequence during translocation
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DOI：
10.1074/jbc.m600882200
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发表时间：
2006-07-07
影响因子：
4.8
通讯作者：
Kowalczykowski, Stephen C.
中科院分区：
文献类型：
--
作者：
Chedin, Frederic;Handa, Naofumi;Kowalczykowski, Stephen C.
枯草芽孢杆菌AddAB酶具有atp依赖性解旋酶和核酸酶活性，导致双链DNA （dsDNA）在易位时解绕和降解。与它的功能对应物大肠杆菌RecBCD酶类似，它也识别并响应特定的DNA序列，称为Chi （Chi）。对chi的识别触发3'-到5'-核酸酶的衰减，这允许产生重组3'-悬垂的单链DNA (ssDNA)，终止于chi。虽然RecBCD酶在chi处短暂停顿，但在易位过程中没有记录到RecBCD与chi的特异性结合。在这里，我们发现AddAB酶在易位过程中会短暂地结合其同源chi序列（chi(Bs):5'- agcg -3'）。AddAB酶与chi（Bs）特异性ssDNA的3'端结合导致保护不被外切酶i降解，这种保护随着时间的推移逐渐降低，在苯酚提取时失去，表明这种结合是非共价的。将AddAB酶添加到已剥离所有蛋白质的加工过的chi（Bs）特异性ssDNA中，不能恢复核酸酶的保护作用，这表明AddAB酶仅在易位过程中以高亲和力与chi（Bs）结合。最后，当易位发生时，携带chi（Bs）的竞争对手ssDNA存在时，仍然观察到chi（Bs）特异性ssDNA的保护作用，表明结合以顺式方式发生。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样酶的生化活性和生物学功能的一般机制。
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.