The invariant glutamate of human PrimPol DxE motif is critical for its Mn-dependent distinctive activities.

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg or Mn, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp, Glu and Asp) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn. Herein, we evidenced that PrimPol Glu contributes to error-prone tolerance of 8oxodG more markedly when both Mg and Mn ions are present. Moreover, Glu was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.