Fig. 7: Proposed mechanism of gap-repair synthesis by Prim–PolC.

Schematic diagram representing the elucidated Prim–PolC catalytic intermediates (yellow boxes) and their positions in the catalytic cycle. (i) Gapped DNA with a 3′-OH and 5′ phosphate is bound by apo Prim–PolC (PDBID: 5OP0). (ii) Upon binding, the 5′ phosphate is located in the binding pocket, and the hydrophobic wedge residues I73/Y74 splay the DNA via insertion between the unpaired bases. (iii) The binary complex then binds an NTP, the DNA splay angle decreases as the upstream DNA docks onto Loop 3 and is tethered via the insertion of the pin residue (N321) that, with P320, disrupts the base pairing at position −2 (PDBID: 6SA0). A process of Synthesis-dependent Template Displacement (STD) allows the ternary complex to be formed. (iv) Nucleotidyl transfer proceeds leaving PPi in the active site, and the extension process pulls the remaining unpaired base into the templating position. The extended upstream duplex disengages from Loop 3 and with an increase in splay angle (PDBID: 6SA1). (v) The gating residue (R179) moves into an open conformation, PPi leaves the active site and is replaced by the next incoming NTP. (vi) The DNA splay angle decreases as the upstream DNA docks onto Loop 3 and is tethered via the insertion of N321 that disrupts the base pairing at position −2. Another ternary complex is formed. (vii) Following nucleotidyl transfer, the upstream duplex disengages from Loop 3, and the downstream DNA disengages from the phosphate-binding pocket, leaving a nick that is then ligated by LigC to complete repair of the DNA.