Biochemical Investigation and Engineering of a Tardigrade X Family DNA Polymerase for Template-Independent DNA Synthesis

The X family of DNA polymerases (PolXs) includes the well-studied mammalian polymerases Polβ, Polλ, Polμ, and terminal deoxynucleotidyl transferase (TdT). The template-independent DNA polymerase activity of TdT has been harnessed for applications in enzymatic de novo DNA synthesis, offering a strategy to overcome the limitations of traditional phosphoramidite-based DNA synthesis methods. Close homologues of the mammalian PolXs are present in other vertebrates, while invertebrate PolXs remain unexplored. In this study, we characterize an invertebrate PolX from the extremotolerant tardigrade Ramazzottius varieornatus (RvPolX), and demonstrate that it possesses modest template-independent DNA polymerase activity, despite limited homology to mammalian PolXs (21% sequence identity with TdT). Through a combination of structural modeling, targeted mutagenesis of active site residues, and high-throughput screening under stringent high salt conditions, we identified a synergistic combination of two mutations (G513A and R522I) that led to a significant increase in activity for the incorporation of all four nucleotides, particularly dATP (∼35-fold), yielding a salt-tolerant polymerase with overall higher activity and substrate promiscuity. Under high salt conditions, the engineered RvPolX displays an activity comparable to TdT and a nucleotide selectivity complementary to TdT. As a template-independent polymerase with a low homology to TdT, RvPolX provides an alternative scaffold for further engineering in various biotechnological applications.