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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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

PrimPol is a recently described DNA polymerase that has the virtue of initiating DNA synthesis. In addition of being a sensu stricto DNA primase, PrimPol's polymerase activity has a large capacity to tolerate different kind of lesions. The different strategies used by PrimPol for DNA damage tolerance are based on its capacity to "read" certain lesions, to skip unreadable lesions, and as an ultimate solution, to restart DNA synthesis beyond the lesion thus acting as a TLS primase. This lesion bypass potential, revised in this article, is strengthened by the preferential use of moderate concentrations of manganese ions as the preferred metal activator. We show here that PrimPol is able to extend RNA primers with ribonucleotides, even when bypassing 8oxoG lesions, suggesting a potential new scenario for PrimPol as a TLS polymerase assisting transcription. We also show that PrimPol displays a high degree of versatility to accept or induce distortions of both primer and template strands, creating alternative alignments based on microhomology that would serve to skip unreadable lesions and to connect separate strands. In good agreement, PrimPol is highly prone to generate indels at short nucleotide repeats. Finally, an evolutionary view of the relationship between translesion synthesis and primase functions is briefly discussed., (Copyright © 2015 Elsevier B.V. All rights reserved.)