The invariant glutamate of human PrimPol DxE motif is critical for its Mn2+-dependent distinctive activities

Abstract 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 2+ or Mn 2+ , 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 114 , Glu 116 and Asp 280 ) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu 116 as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn 2+ . Herein, we evidenced that PrimPol Glu 116 contributes to error-prone tolerance of 8oxodG more markedly when both Mg 2+ and Mn 2+ ions are present. Moreover, Glu 116 was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn 2+ 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 116 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 2+ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.