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Publication Abstract from PubMed

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.

Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase.,Lelyveld VS, Zhang W, Szostak JW Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7276-7283. doi:, 10.1073/pnas.1922400117. Epub 2020 Mar 18. PMID:32188786^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.