Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.
Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder.
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DOI：
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发表时间：
2003
期刊：
影响因子：
56.9
通讯作者：
Antoine M. van Oijen;P. Blainey;D. J. Crampton;C. Richardson;T. Ellenberger;X. Xie
中科院分区：
文献类型：
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作者：
Antoine M. van Oijen;P. Blainey;D. J. Crampton;C. Richardson;T. Ellenberger;X. Xie
我们使用了基于流动拉伸DNA的多重方法来监测单个噬菌体λ核酸外切酶分子对噬菌体DNA的酶消化。同时观察到的多个单分子轨迹的统计分析表明，催化速率取决于底物DNA的局部碱基含量。通过将单分子动力学与氢键和碱基堆积的自由能相关联，我们确定了从DNA中熔化碱基是催化循环中的限速步骤。催化速率也表现出大的波动独立的序列，我们归因于构象变化的酶-DNA复合物。
We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.