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Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

Tushar Aggarwal, William A. Hansen, Jonathan Hong, View ORCID ProfileAbir Ganguly, View ORCID ProfileDarrin M. York, View ORCID ProfileSagar D. Khare, View ORCID ProfileEnver Cagri Izgu
doi: https://doi.org/10.1101/2021.12.28.474375
Tushar Aggarwal
1Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ 08854, USA
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William A. Hansen
2Institute for Quantitative Biomedicine, Rutgers University, New Brunswick, NJ 08854, USA
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Jonathan Hong
1Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ 08854, USA
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Abir Ganguly
2Institute for Quantitative Biomedicine, Rutgers University, New Brunswick, NJ 08854, USA
3Laboratory for Biomolecular Simulation Research, Rutgers University, New Brunswick, NJ 08854, USA
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  • ORCID record for Abir Ganguly
Darrin M. York
1Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ 08854, USA
2Institute for Quantitative Biomedicine, Rutgers University, New Brunswick, NJ 08854, USA
3Laboratory for Biomolecular Simulation Research, Rutgers University, New Brunswick, NJ 08854, USA
4Cancer Institute of New Jersey, Rutgers University, New Brunswick, NJ 08901, USA
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Sagar D. Khare
1Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ 08854, USA
2Institute for Quantitative Biomedicine, Rutgers University, New Brunswick, NJ 08854, USA
4Cancer Institute of New Jersey, Rutgers University, New Brunswick, NJ 08901, USA
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  • For correspondence: khare@chem.rutgers.edu ec.izgu@rutgers.edu
Enver Cagri Izgu
1Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ 08854, USA
4Cancer Institute of New Jersey, Rutgers University, New Brunswick, NJ 08901, USA
5Rutgers Center for Lipid Research and New Jersey Institute for Food, Nutrition, and Health, Rutgers University, New Brunswick, NJ 08901, USA
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  • ORCID record for Enver Cagri Izgu
  • For correspondence: khare@chem.rutgers.edu ec.izgu@rutgers.edu
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ABSTRACT

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9°N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3’→P5’ phosphoramidate linkages in the presence of 3’-amino-2’,3’-dideoxynucleoside 5’-triphosphate (3’-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9°N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3’-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9°N-NRQS) that incorporated 3’-NH2-ddNTPs into a 3’-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 29, 2021.
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Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign
Tushar Aggarwal, William A. Hansen, Jonathan Hong, Abir Ganguly, Darrin M. York, Sagar D. Khare, Enver Cagri Izgu
bioRxiv 2021.12.28.474375; doi: https://doi.org/10.1101/2021.12.28.474375
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Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign
Tushar Aggarwal, William A. Hansen, Jonathan Hong, Abir Ganguly, Darrin M. York, Sagar D. Khare, Enver Cagri Izgu
bioRxiv 2021.12.28.474375; doi: https://doi.org/10.1101/2021.12.28.474375

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