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.