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

Patricia A Calvo,Guillermo Sastre-Moreno,Cristina Perpiñá,Susana Guerra,María I Martínez-Jiménez,Luis Blanco

doi:10.1016/j.dnarep.2019.03.006

Patricia A Calvo, Guillermo Sastre-Moreno + Show 4 more

Open Access

https://doi.org/10.1016/j.dnarep.2019.03.006

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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.

Highlights

In silico analyses of the primary sequence of human PrimPol indicate that it belongs to the archaeo-eukaryotic superfamily of primases (AEP) containing a diverged version of the RNA recognition motif (RRM) fold [1]
The AEP superfamily is highly heterogeneous as it encompasses conventional primases, PrimPols, and even RNA polymerases specialized in the nonhomologous end-joining (NHEJ) of double-strand breaks (DSBs) [2,3]
The DxD motif is present in most members of the AEP superfamily including the conventional human primase Prim1, viral and bacterial and phage primases, and in bacterial AEPs involved in NHEJ, as MtPolDom (Fig. 1A)

Summary

Introduction

In silico analyses of the primary sequence of human PrimPol indicate that it belongs to the archaeo-eukaryotic superfamily of primases (AEP) containing a diverged version of the RNA recognition motif (RRM) fold [1]. PrimPol harbors the conserved A, B, C motifs of the AEP superfamily and a C-terminal subdomain containing a Zincfinger motif, similar to that of herpesvirus UL52 primase and other AEPlike enzymes, which was shown to be essential for its primase activity [4,5,6]. PrimPol is the first DNA primase characterized in human cells, as it is able to start DNA chains with deoxynucleotides, with a unique active site for both DNA primase and DNA polymerase activities [7,8,9]. PrimPol primase activity was demonstrated to be relevant for reinitiating stalled forks after UV damage during nuclear DNA replication [4]. PrimPol primase activity was shown to be required during mitochondrial DNA replication, by reinitiating synthesis after UV damage or in the presence of chain-terminating nucleotides [7,13]

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