Thermococcus sp. 9°N DNA polymerase exhibits 3′-esterase activity that can be harnessed for DNA sequencing

Shiuan Woei LinWu, Yu Hsuan Tu, Ting Yueh Tsai, Manuel Maestre-Reyna, Mu Sen Liu, Wen Jin Wu, Jyun Yuan Huang, Hung Wen Chi, Wei Hsin Chang, Chung Fan Chiou, Andrew H.J. Wang, Johnsee Lee, Ming Daw Tsai

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3 引文 斯高帕斯(Scopus)

摘要

It was reported in 1995 that T7 and Taq DNA polymerases possess 3′-esterase activity, but without follow-up studies. Here we report that the 3′-esterase activity is intrinsic to the Thermococcus sp. 9°N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3′-ester with a fluorophore. We first show that 3′-esterified dNTP can be incorporated into a template-primer DNA, and solve the crystal structures of the reaction intermediates and products. Then we show that the reaction can occur continuously, modulated by active site residues Tyr409 and Asp542. Finally, we use 5′-FAM-labeled primer and esterified dNTP with a dye to show that the reaction can proceed to ca. 450 base pairs, and that the intermediates of many individual steps can be identified. The results demonstrate the feasibility of a 3′-editing based DNA sequencing method that could find practical applications after further optimization.

原文英語
文章編號224
期刊Communications Biology
2
發行號1
DOIs
出版狀態已發佈 - 12月 1 2019
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ASJC Scopus subject areas

  • 醫藥(雜項)
  • 生物化學、遺傳與分子生物學 (全部)
  • 農業與生物科學 (全部)

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