The invariant glutamate of human PrimPol DxE motif is critical for its Mn²⁺-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 Mg²⁺ or Mn²⁺, 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 (Asp¹¹⁴, Glu¹¹⁶ and Asp²⁸⁰) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu¹¹⁶ as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn²⁺. Herein, we evidenced that PrimPol Glu¹¹⁶ contributes to error-prone tolerance of 8oxodG more markedly when both Mg²⁺ and Mn²⁺ ions are present. Moreover, Glu¹¹⁶ was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn²⁺ 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 Glu¹¹⁶ 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 Mn²⁺ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.

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