The invariant glutamate of human PrimPol DxE motif is critical for its Mn 2+ -dependent distinctive activities.

•Human PrimPol requires D114, E116 and D280 as activating metal ligands.•PrimPol metal ligands are irrelevant for ssDNA template binding.•PrimPol:ssDNA:dNTP complex formation requires Mn2+ and the three metal ligands.•Glu116 contributes to error-prone tolerance of 8oxodG.•Glu116 enhances Mn2+-dependent PrimPol primase and polymerase activities.