Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 7857, Konstanz, Germany.
Department of Biology and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457, Konstanz, Germany.
Angew Chem Int Ed Engl. 2019 Apr 8;58(16):5457-5461. doi: 10.1002/anie.201900315. Epub 2019 Mar 8.
Archaeal B-family DNA polymerases (DNA pols) are the driving force of cutting-edge biotechnological applications like next-generation sequencing. The acceptance of chemically modified nucleotides by DNA pols is key to these technologies. Until now, no structural data have been available for these DNA pols in complex with modified substrates, which could build the basis for understanding interactions between the enzyme and the chemically modified nucleotide and for the further development of next-generation nucleotides. For the first time, we crystallized an exonuclease-deficient variant of the wild-type B-family KOD DNA pol with a modified nucleotide in a closed, ternary complex. We also crystalized the A-family DNA pol KlenTaq with the same nucleotide. The reported structural data reveal how the protein and the DNA modulate two distinct conformations of the appended moiety in the A- and B-family DNA pols and how these influence the processing of the modified nucleotide. Overall, this study provides first insight into the interplay between B-family DNA pols and relevant modified substrates.
古菌 B 族 DNA 聚合酶（DNA pols）是下一代测序等前沿生物技术应用的推动力。DNA pols 对化学修饰核苷酸的接受能力是这些技术的关键。到目前为止，还没有这些与修饰底物形成复合物的 DNA pols 的结构数据，这些数据可以为理解酶与化学修饰核苷酸之间的相互作用以及下一代核苷酸的进一步发展奠定基础。我们首次结晶了带有封闭三元复合物中修饰核苷酸的野生型 B 族 KOD DNA pol 的缺少外切酶的变体，同时还结晶了 A 族 DNA pol KlenTaq 与相同核苷酸的复合物。报道的结构数据揭示了蛋白质和 DNA 如何调节 A 族和 B 族 DNA pol 中附加部分的两种不同构象，以及这些构象如何影响修饰核苷酸的处理。总的来说，这项研究首次深入了解了 B 族 DNA pols 与相关修饰底物之间的相互作用。
