6US5

Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase.

(2020) Proc Natl Acad Sci U S A 117: 7276-7283

• PubMed: 32188786 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1073/pnas.1922400117
• Primary Citation of Related Structures:  
  6UR2, 6UR4, 6UR9, 6US5


• PubMed Abstract: 
  

  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.

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Organizational Affiliation: 

Department of Molecular Biology, Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114.