The recent discovery of Drt3b, a bacterial antiphage reverse transcriptase that synthesizes poly(AC) DNA template-independently via a protein pseudotemplate mechanism (Glu26 and Arg253), provides a conceptual framework for engineering analogous activity into human enzymes 4. Here we describe the computational design and molecular dynamics (MD) validation of PrimPol-T6 v9a, a variant of human PrimPol (UniProt Q96LW4, 560 aa) bearing four mutations (S160R, K165R, F166Y, S167R) in and adjacent to Motif II of the active site. Chai-1 structure prediction with distance restraints, followed by 20 ns MD simulations with catalytic Mg2+ ions (GROMACS 2025.1, amber99sb-ildn, 310 K), demonstrated stable positioning of single-stranded telomeric DNA (TTAGGGTTAGGGTTAGGG) in the redesigned active site: Arg167 maintained hydrogen-bond contact with DNA in 99.7% of simulation frames (mean 2.77 Å), while catalytic Mg2+ coordination by Asp114 was stable throughout (mean 1.87 A, 100% occupancy). A critical control simulation - v2 (S167R + K165R only) with Mg2+ - showed 0.0% H-bond occupancy (mean 5.54 Å), demonstrating that Mg2+ without the four engineered mutations is insufficient and even counterproductive. The four mutations are therefore necessary and the improvement in v9a reflects genuine synergy between the redesigned active site and catalytic Mg2+. Nine rounds of iterative design identified S160R as a critical fourth mutation, discoverable only through Chai-1 with distance restraints rather than unconstrained AlphaFold3. Beyond telomere biology, these results establish proof-of-concept for Programmable Protein-Templated DNA Synthesis - PPTDS (used in this article; and Programmable Protein-Templated Nucleic Acid Synthesis (PPTNAS) for a wider context of polymerase scaffolds), a new paradigm with broad implications for molecular biotechnology, synthetic biology, and regenerative medicine.
Kukier et al. (Sun,) studied this question.