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

DNA Repair (Amst). 2019 May;77:65-75. doi: 10.1016/j.dnarep.2019.03.006. Epub 2019 Mar 14.

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 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.

Keywords: Catalytic residues; Metal cofactor; Polymerase; Pre-ternary complex; Primase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Hydroxy-2'-Deoxyguanosine / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • DNA Primase / chemistry*
  • DNA Primase / genetics
  • DNA Primase / metabolism*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Glutamic Acid*
  • Humans
  • Ligands
  • Manganese / metabolism*
  • Models, Molecular
  • Multifunctional Enzymes / chemistry*
  • Multifunctional Enzymes / genetics
  • Multifunctional Enzymes / metabolism*
  • Point Mutation
  • Protein Multimerization
  • Protein Structure, Quaternary

Substances

  • Deoxyadenine Nucleotides
  • Ligands
  • Multifunctional Enzymes
  • Glutamic Acid
  • Manganese
  • 8-Hydroxy-2'-Deoxyguanosine
  • DNA Primase
  • PrimPol protein, human
  • DNA-Directed DNA Polymerase
  • 2'-deoxyadenosine triphosphate