Structural basis for TNA synthesis by an engineered TNA polymerase

• Published in: Nature communications Vol. 8; no. 1; pp. 1810 - 11
• Main Authors: Chim, Nicholas, Shi, Changhua, Sau, Sujay P., Nikoomanzar, Ali, Chaput, John C.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 27.11.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 631/45/535/1266; 631/92/610; 631/92/612/1229; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Biological Evolution; Catalysis; Catalytic Domain; Chemical synthesis; Crystal structure; Crystallography, X-Ray; Deoxyribonucleic acid; DNA; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Humanities and Social Sciences; Inspection; multidisciplinary; Mutation; Nucleic Acid Conformation; Nucleic acids; Nucleic Acids - biosynthesis; Nucleic Acids - chemistry; Nucleosides - chemistry; Nucleosides - metabolism; Protein Engineering; Science; Science & Technology - Other Topics; Science (multidisciplinary)
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-017-02014-0

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-( l )-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants. The laboratory-evolved polymerase Kod-RI catalyzes α-L-threose nucleic acid (TNA) synthesis. Here, the authors present Kod-RI crystal structures that give insights into how TNA triphosphates are selected and extended in a template-dependent manner, which will help to engineer improved TNA polymerases for synthetic genetics applications.