Department of Pharmaceutical Sciences, University of California, Irvine, CA, USA.
Department of Chemical and Biomolecular Engineering, University of California, Irvine, CA, USA.
Nat Commun. 2021 May 11;12(1):2641. doi: 10.1038/s41467-021-22937-z.
The mechanism of DNA synthesis has been inferred from static structures, but the absence of temporal information raises longstanding questions about the order of events in one of life's most central processes. Here we follow the reaction pathway of a replicative DNA polymerase using time-resolved X-ray crystallography to elucidate the order and transition between intermediates. In contrast to the canonical model, the structural changes observed in the time-lapsed images reveal a catalytic cycle in which translocation precedes catalysis. The translocation step appears to follow a push-pull mechanism where the O-O1 loop of the finger subdomain acts as a pawl to facilitate unidirectional movement along the template with conserved tyrosine residues 714 and 719 functioning as tandem gatekeepers of DNA synthesis. The structures capture the precise order of critical events that may be a general feature of enzymatic catalysis among replicative DNA polymerases.
DNA 合成的机制是从静态结构中推断出来的，但由于缺乏时间信息，关于生命中最核心过程之一的事件顺序的长期问题仍然存在。在这里，我们使用时间分辨 X 射线晶体学来追踪复制 DNA 聚合酶的反应途径，以阐明中间体之间的顺序和转变。与典型模型相比，时程图像中观察到的结构变化揭示了一个催化循环，其中转位先于催化。转位步骤似乎遵循推挽机制，其中指状亚基的 O-O1 环充当棘爪，以促进沿模板的单向运动，保守的酪氨酸残基 714 和 719 作为 DNA 合成的串联门卫。这些结构捕捉到了关键事件的精确顺序，这可能是复制 DNA 聚合酶中酶催化的一个普遍特征。
