Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase–nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5′-ended strand and its slower RecB helicase on the 3′-ended strand. At Chi hotspots (5′ GCTGGTGG 3′), RecB’s nuclease cuts the 3′-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB’s ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

中文翻译：

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RecBCD 解旋酶核酸酶对 Chi 热点激活状态的小分子敏化。


协调复杂酶的多种活动对于生命至关重要，包括转录、复制和修复 DNA。细菌 RecBCD 解旋酶-核酸酶必须协调 DNA 解旋和切割以修复断裂的 DNA。从 DNA 末端开始，RecBCD 使用 5' 端链上的快速 RecD 解旋酶和 3' 端链上的较慢 RecB 解旋酶解旋 DNA。在 Chi 热点 (5' GCTGGTGG 3')，RecB 的核酸酶切割 3' 端链并将 RecA 链交换蛋白加载到其上。我们报道了一种小分子 NSAC1003（一种磺基三唑并苯并咪唑），通过敏化 RecBCD 来模拟 Chi 位点，在与 Chi 无关的位置切割 DNA 一定百分比的 DNA 底物长度。该百分比随着 NSAC1003 浓度的增加而降低。我们的数据表明，相对于 RecD，NSAC1003 会减慢 RecB 的速度，并且当领先的解旋酶 RecD 在 DNA 末端停止时，它会使其对 DNA 切割变得敏感。之前描述的两种 RecB ATP 结合位点发生改变的 RecBCD 突变体也具有此特性，但未抑制的野生型 RecBCD 缺乏此特性。 ATP和NSAC1003具有竞争力；计算将 NSAC1003 对接至 RecB 的 ATP 结合位点，表明 NSAC1003 直接作用于 RecB。 NSAC1003 将有助于阐明 RecBCD-Chi 调节和 DNA 修复的分子机制。类似的研究可以帮助阐明其他在染色体位点上具有协调活性的 DNA 酶。