LAUSR

Significance Life on Earth depends on polymerases. These enzymes copy genetic information to produce the DNA and RNA strands at the core of the central dogma. Polymerases act by forming phosphodiester linkages to produce polynucleotide strands. While synthetic chemistry can generate a broad range of alternative genetic materials with unnatural linkages, polymerases have so far been limited to forming O-P bonds. Here, we show that, in fact, unnatural N-P bonds can also be formed by a modified DNA polymerase. This template-directed activity generates complementary strands linked by phosphoramidate (NP) esters, an alternative backbone linkage only known to exist in the laboratory. The emergence of NP-DNA polymerase activity implies the biochemical plausibility of alternative central dogmas for cellular life. 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 Ca2+ rather than Mg2+, 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.