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Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase
All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservati...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
National Academy of Sciences
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132125/ https://www.ncbi.nlm.nih.gov/pubmed/32188786 http://dx.doi.org/10.1073/pnas.1922400117 |
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author | Lelyveld, Victor S. Zhang, Wen Szostak, Jack W. |
author_facet | Lelyveld, Victor S. Zhang, Wen Szostak, Jack W. |
author_sort | Lelyveld, Victor S. |
collection | PubMed |
description | All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3′-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3′-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3′→P5′ phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3′-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca(2+) rather than Mg(2+), the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA. |
format | Online Article Text |
id | pubmed-7132125 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | National Academy of Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-71321252020-04-09 Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase Lelyveld, Victor S. Zhang, Wen Szostak, Jack W. Proc Natl Acad Sci U S A Biological Sciences All known polymerases copy genetic material by catalyzing phosphodiester bond formation. This highly conserved activity proceeds by a common mechanism, such that incorporated nucleoside analogs terminate chain elongation if the resulting primer strand lacks a terminal hydroxyl group. Even conservatively substituted 3′-amino nucleotides generally act as chain terminators, and no enzymatic pathway for their polymerization has yet been found. Although 3′-amino nucleotides can be chemically coupled to yield stable oligonucleotides containing N3′→P5′ phosphoramidate (NP) bonds, no such internucleotide linkages are known to occur in nature. Here, we report that 3′-amino terminated primers are, in fact, slowly extended by the DNA polymerase from B. stearothermophilus in a template-directed manner. When its cofactor is Ca(2+) rather than Mg(2+), the reaction is fivefold faster, permitting multiple turnover NP bond formation to yield NP-DNA strands from the corresponding 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphates. A single active site mutation further enhances the rate of NP-DNA synthesis by an additional 21-fold. We show that DNA-dependent NP-DNA polymerase activity depends on conserved active site residues and propose a likely mechanism for this activity based on a series of crystal structures of bound complexes. Our results significantly broaden the catalytic scope of polymerase activity and suggest the feasibility of a genetic transition between native nucleic acids and NP-DNA. National Academy of Sciences 2020-03-31 2020-03-18 /pmc/articles/PMC7132125/ /pubmed/32188786 http://dx.doi.org/10.1073/pnas.1922400117 Text en Copyright © 2020 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/ https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) . |
spellingShingle | Biological Sciences Lelyveld, Victor S. Zhang, Wen Szostak, Jack W. Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase |
title | Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase |
title_full | Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase |
title_fullStr | Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase |
title_full_unstemmed | Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase |
title_short | Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase |
title_sort | synthesis of phosphoramidate-linked dna by a modified dna polymerase |
topic | Biological Sciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132125/ https://www.ncbi.nlm.nih.gov/pubmed/32188786 http://dx.doi.org/10.1073/pnas.1922400117 |
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