Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme
Bipolar DNA translocation contributes to highly processive DNA unwinding by RecBCD enzyme
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DOI：
10.1074/jbc.m505520200
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发表时间：
2005-11-04
影响因子：
4.8
通讯作者：
Kowalczykowski, SC
中科院分区：
文献类型：
--
作者：
Dillingham, MS;Webb, MR;Kowalczykowski, SC
我们最近证明了RecBCD酶是一种双极DNA解旋酶，它采用两个极性相反的单链DNA马达来驱动双链DNA的易位和解旋。我们假设，这种组织可以解释由RecBCD酶催化的DNA解旋的异常高的速率和持续性。使用停流染料置换分析解旋活性，我们测试这一想法通过分析突变体RecBCD酶，其中两个解旋酶马达中的任一个通过诱变失活。与野生型RecBCD酶一样，这两种突变蛋白在缺乏ATP的情况下保持与钝双链DNA末端紧密结合的能力。然而，RecB马达缺陷型酶的正向易位率仅与野生型酶的30%相似，而RecD马达缺陷型酶的正向易位率与野生型酶的50%相似。更重要的是，易位的持续合成能力大大降低，分别为每种突变酶的25倍和6倍。尽管保留了结合平端dsDNA的能力，但RecB突变体酶已经失去了解旋DNA的能力，除非底物含有短的5 '端单链DNA突出端。单链DNA马达的起始复合物的架构，这一观察的后果进行了讨论。
We recently demonstrated that the RecBCD enzyme is a bipolar DNA helicase that employs two single-stranded DNA motors of opposite polarity to drive translocation and unwinding of duplex DNA. We hypothesized that this organization may explain the exceptionally high rate and processivity of DNA unwinding catalyzed by the RecBCD enzyme. Using a stopped-flow dye displacement assay for unwinding activity, we test this idea by analyzing mutant RecBCD enzymes in which either of the two helicase motors is inactivated by mutagenesis. Like the wild-type RecBCD enzyme, the two mutant proteins maintain the ability to bind tightly to blunt duplex DNA ends in the absence of ATP. However, the rate of forward translocation for the RecB motor-defective enzyme is only similar to 30% of the wild-type rate, whereas for the RecD motor-defective enzyme, it is similar to 50%. More significantly, the processivity of translocation is substantially reduced by similar to 25- and 6-fold for each mutant enzyme, respectively. Despite retaining the capacity to bind blunt dsDNA, the RecB-mutant enzyme has lost the ability to unwind DNA unless the substrate contains a short 5'-terminated single-stranded DNA overhang. The consequences of this observation for the architecture of the single-stranded DNA motors in the initiation complex are discussed.