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Título: | The invariant glutamate of human PrimPol DxE motif is critical for its Mn2+-dependent distinctive activities |
Autor: | Calvo, Patricia A.; Sastre-Moreno, Guillermo; Perpiña, Cristina; Guerra, Susana; Martínez-Jiménez, María I. CSIC ORCID ; Blanco, Luis CSIC ORCID | Palabras clave: | Primare Polymerase Metal cofactor Catalytic residues Pre-ternary complex |
Fecha de publicación: | 14-mar-2019 | Editor: | Elsevier | Citación: | DNA Repair | Resumen: | 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 PrimPolmediated 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 errorprone 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. | Versión del editor: | https://doi.org/10.1016/j.dnarep.2019.03.006 | URI: | http://hdl.handle.net/10261/246714 | ISSN: | 1568-7856 |
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