Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair
Molecular wrench activity of DNA helicases: Keys to modulation of rapid kinetics in DNA repair
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DOI：
10.1002/pro.4815
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发表时间：
2023-12-01
期刊：
影响因子：
8
通讯作者：
Slinker，Jason D.
中科院分区：
文献类型：
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作者：
Wettasinghe，Ashan P.;Seifi，Melodee O.;Slinker，Jason D.
DNA 解旋酶活性对于重组、复制、转录、翻译和修复等重要的 DNA 代谢过程至关重要。最近，与来自 Sulfolobus tokodaii、StXPB1 和 StXPB2 的较慢的传统解旋酶相比，在古生球菌 fulgidushelicase (AfXPB) 中观察到出乎意料的、快速的指数 ATP 刺激 DNA 解旋速率。这种不寻常的快速活性表明，在从开放构象转变为闭合构象的过程中，AfXPB 在双链体结构上施加的扭矩产生了“分子扳手”机制。然而，还有很多事情有待理解。在这里，我们通过氧化还原活性 DNA 单层的电化学测定，研究了 StXPB2 和 AfXPB 的 DNA 解旋酶结合和 ATP 刺激动力学的浓度依赖性，以及它们在 Bax1 复合物中的结合和活性。 StXPB2 ATP 刺激活性在 8 至 200nM 范围内与浓度无关。出乎意料的是，AfXPB 活性在此范围内呈浓度依赖性，指数速率常数从浓度大于 20nM 的数秒到较低浓度的数千秒不等。在 20nM 时，信号会发生快速指数衰减，线性反转，然后以较慢的指数衰减恢复。 AfXPB 活性随其浓度变化的这种变化被合理化为快速分子扳手和较慢的常规解旋酶模式之间的交叉。 AfXPB-Bax1 抑制快速活性，而 StXPB2-Bax1 复合物在较高浓度下诱导快速动力学。这些配合物的晶体结构使该活性合理化。这些发现阐明了控制分子扳手活动的不同物理模型，以提高对 DNA 修复关键因素的生物学洞察。
DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP‐stimulated DNA unwinding rate was observed from anArchaeoglobus fulgidushelicase (AfXPB) as compared to the slower conventional helicases fromSulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a “molecular wrench” mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP‐stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox‐active DNA monolayers. StXPB2 ATP‐stimulated activity is concentration‐independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration‐dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB‐Bax1 inhibits rapid activity, whereas the StXPB2‐Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.