Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.
Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.
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DOI：
10.1093/nar/gkv204
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发表时间：
2015-04-20
影响因子：
14.9
通讯作者：
Ibarra B
中科院分区：
文献类型：
--
作者：
Morin JA;Cao FJ;Lázaro JM;Arias-Gonzalez JR;Valpuesta JM;Carrascosa JL;Salas M;Ibarra B
在DNA复制过程中，复制型聚合酶沿着DNA模板以离散的机械步骤移动。为了解决化学循环如何耦合到酶的机械运动，在这里，我们使用光镊研究单个噬菌体Phi 29 DNA聚合酶在进行性DNA复制过程中的易位机制。我们确定的主要动力学参数的核苷酸掺入周期和它们依赖于外部负载和核苷酸（dNTP）浓度。这些数据与易位的动力冲程模型不一致，而是支持DNA复制过程中化学催化和机械易位之间的松耦合机制。根据这种机制，DNA聚合酶通过在dNTP/PPi游离状态和dNTP/PPi结合状态之间交替工作，dNTP/PPi游离状态在易位前和易位后状态之间热扩散，dNTP/PPi结合状态使易位后状态稳定。我们展示了聚合酶如何使用这种热棘轮机制来产生对大的相对负载（150 pN）的工作。
During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).
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