Raper Austin T, Suo Zucai
Department of Chemistry and Biochemistry, The Ohio State University , Columbus, Ohio 43210, United States.
Ohio State Biochemistry Program, The Ohio State University , Columbus, Ohio 43210, United States.
Biochemistry. 2016 Oct 18;55(41):5832-5844. doi: 10.1021/acs.biochem.6b00878. Epub 2016 Oct 10.
DNA polymerases catalyze DNA synthesis through a stepwise kinetic mechanism that begins with binding to DNA, followed by selection, binding, and incorporation of a nucleotide into an elongating primer. It is hypothesized that subtle active site adjustments in a polymerase to align reactive moieties limit the rate of correct nucleotide incorporation. DNA damage can impede this process for many DNA polymerases, causing replication fork stalling, genetic mutations, and potentially cell death. However, specialized Y-family DNA polymerases are structurally evolved to efficiently bypass DNA damage in vivo, albeit at the expense of replication fidelity. Dpo4, a model Y-family polymerase from Sulfolobus solfataricus, has been well-studied kinetically, structurally, and computationally, which yielded a mechanistic understanding of how the Y-family DNA polymerases achieve their unique catalytic properties. We previously employed a real-time Förster resonance energy transfer (FRET) technique to characterize the global conformational motions of Dpo4 during DNA binding as well as nucleotide binding and incorporation by monitoring changes in distance between sites on the polymerase and DNA, and even between domains of Dpo4. Here, we extend the utility of our FRET methodology to observe conformational transitions within individual domains of Dpo4 during DNA binding and nucleotide incorporation. The results of this novel, intradomain FRET approach unify findings from many studies to fully clarify the complex DNA binding mechanism of Dpo4. Furthermore, intradomain motions in the Finger domain during nucleotide binding and incorporation, for the first time, report on the rate-limiting step of a single-nucleotide addition catalyzed by Dpo4.
DNA聚合酶通过逐步的动力学机制催化DNA合成，该机制始于与DNA结合，随后是核苷酸的选择、结合以及掺入正在延长的引物中。据推测，聚合酶中微妙的活性位点调整以对齐反应性基团会限制正确核苷酸掺入的速率。DNA损伤会阻碍许多DNA聚合酶的这一过程，导致复制叉停滞、基因突变，并可能导致细胞死亡。然而，专门的Y家族DNA聚合酶在结构上经过进化，能够在体内有效地绕过DNA损伤，尽管这是以复制保真度为代价的。Dpo4是来自嗜热栖热菌的一种典型Y家族聚合酶，已在动力学、结构和计算方面得到了充分研究，从而对Y家族DNA聚合酶如何实现其独特的催化特性有了机理上的理解。我们之前采用实时Förster共振能量转移（FRET）技术，通过监测聚合酶与DNA上位点之间甚至Dpo4结构域之间距离的变化，来表征Dpo4在DNA结合以及核苷酸结合和掺入过程中的全局构象运动。在这里，我们扩展了FRET方法的应用，以观察Dpo4在DNA结合和核苷酸掺入过程中各个结构域内的构象转变。这种新颖的结构域内FRET方法的结果统一了许多研究的发现，以充分阐明Dpo4复杂的DNA结合机制。此外，在核苷酸结合和掺入过程中，手指结构域内的结构域内运动首次揭示了Dpo4催化的单核苷酸添加的限速步骤。
