Frevert Zach, Reusch Devin T, Gildenberg Melissa S, Jordan Sarah M, Ryan Benjamin J, Freudenthal Bret D, Washington M Todd
Department of Biochemistry and Molecular Biology, University of Iowa College of Medicine, Iowa City, IA, USA.
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, USA.
Nat Commun. 2025 Jul 1;16(1):5979. doi: 10.1038/s41467-025-61245-8.
Translesion synthesis polymerases efficiently incorporate nucleotides opposite DNA lesions. Pol ι, for example, bypasses minor-groove and exocyclic purine adducts. Conventional X-ray crystallography showed that this enzyme incorporates nucleotides by forming Hoogsteen base pairs with the incoming nucleotide rather than Watson-Crick base pairs. While this revealed the structural basis of nucleotide selection during nucleotide binding, it did not allow the visualization of the process of phosphodiester bond formation or the detection of reaction intermediates that form during nucleotide incorporation. Here, we use a combination of time-lapse crystallography and molecular dynamics simulations to examine the mechanism of pol ι-catalyzed nucleotide incorporation. We show that this enzyme maintains Hoogsteen base pairing with the incoming dNTP during the entire reaction. We also show that pol ι possesses a pyrophosphatase activity that generates two monophosphates within its active site. Our findings provide insights into the features of pol ι's active site that allow it to translocate along DNA and catalyze processive DNA synthesis.
跨损伤合成聚合酶能够有效地在DNA损伤位点对面掺入核苷酸。例如，聚合酶ι能够绕过小沟和环外嘌呤加合物。传统的X射线晶体学研究表明，该酶通过与进入的核苷酸形成Hoogsteen碱基对而非沃森-克里克碱基对来掺入核苷酸。虽然这揭示了核苷酸结合过程中核苷酸选择的结构基础，但它无法可视化磷酸二酯键形成的过程，也无法检测核苷酸掺入过程中形成的反应中间体。在这里，我们结合使用延时晶体学和分子动力学模拟来研究聚合酶ι催化的核苷酸掺入机制。我们发现，该酶在整个反应过程中与进入的dNTP保持Hoogsteen碱基配对。我们还发现，聚合酶ι具有焦磷酸酶活性，能够在其活性位点产生两个单磷酸。我们的研究结果为聚合酶ι活性位点的特征提供了见解，这些特征使其能够沿着DNA移位并催化连续的DNA合成。
