A cancer-associated point mutation disables the steric gate of human PrimPol

Luis Blanco-Franco,Luis Blanco,Alberto Díaz-Talavera,Susana Guerra,Maria I Martínez-Jiménez,Marcos Díaz,Juan Méndez,Patricia A Calvo,Guillermo Sastre-Moreno,Daniel González-Acosta

doi:10.1038/s41598-018-37439-0

Abstract

PrimPol is a human primase/polymerase specialized in re-starting stalled forks by repriming beyond lesions such as pyrimidine dimers, and replication-perturbing structures including G-quadruplexes and R-loops. Unlike most conventional primases, PrimPol proficiently discriminates against ribonucleotides (NTPs), being able to start synthesis using deoxynucleotides (dNTPs), yet the structural basis and physiological implications for this discrimination are not understood. In silico analyses based on the three-dimensional structure of human PrimPol and related enzymes enabled us to predict a single residue, Tyr100, as the main effector of sugar discrimination in human PrimPol and a change of Tyr100 to histidine to boost the efficiency of NTP incorporation. We show here that the Y100H mutation profoundly stimulates NTP incorporation by human PrimPol, with an efficiency similar to that for dNTP incorporation during both primase and polymerase reactions in vitro. As expected from the higher cellular concentration of NTPs relative to dNTPs, Y100H expression in mouse embryonic fibroblasts and U2OS osteosarcoma cells caused enhanced resistance to hydroxyurea, which decreases the dNTP pool levels in S-phase. Remarkably, the Y100H PrimPol mutation has been identified in cancer, suggesting that this mutation could be selected to promote survival at early stages of tumorigenesis, which is characterized by depleted dNTP pools.

Highlights

Incorporation of ribonucleoside triphosphates (NTPs) onto DNA is generally considered harmful, because the persistence of ribonucleoside monophosphates (NMPs) in the DNA is associated with several potential problems: (1) NMPs are non-canonical templates for DNA replication[1,2,3]; (2) the assembly of nucleosomes is impeded by the presence of NMPs4; (3) NMPs embedded in the DNA are more prone to hydrolysis than deoxynucleosides monophosphates and render the DNA backbone more labile[5]; and (4) a single NMP embedded in the DNA duplex can result in helix perturbation and can alter protein recognition and binding[6,7]
Pyrococcus furiosus p41 (Pfu-p41), an archeal primase/polymerase[31,32,33], was chosen as an AEP with preference for dNTPs, similar to human PrimPol[34]; in turn, Mycobacterium tuberculosis PolDom (MtPolDom), the polymerase domain in Ligase D, a multi-domain protein specialized in non-homologous end joining (NHEJ)[35,36,37], was chosen as an AEP with preference for NTP insertion
The fact that tyrosines are bulkier than histidines supports a role in excluding NTPs by steric hindrance rather than stabilizing these substrates in the active site, which could explain the difference in sugar selectivity between human PrimPol/Pfu-p41 and MtPolDom

Summary

Introduction

Incorporation of ribonucleoside triphosphates (NTPs) onto DNA is generally considered harmful, because the persistence of ribonucleoside monophosphates (NMPs) in the DNA is associated with several potential problems: (1) NMPs are non-canonical templates for DNA replication[1,2,3]; (2) the assembly of nucleosomes is impeded by the presence of NMPs4; (3) NMPs embedded in the DNA are more prone to hydrolysis than deoxynucleosides monophosphates (dNMPs) and render the DNA backbone more labile[5]; and (4) a single NMP embedded in the DNA duplex can result in helix perturbation and can alter protein recognition and binding[6,7]. Recent studies demonstrated that despite their ability to discriminate against NTPs, replicases incorporate these substrates at strikingly high rates in vivo (e.g. around 1 per 1 kb in the case of yeast Polε) because of their high cellular concentration[11] This incorporation of NTPs is not necessarily hazardous as single embedded NMPs are efficiently removed by the ribonucleotide excision repair pathway[12], which is initiated by RNase H2, an enzyme essential to preserve genomic stability in mammals[13]. We have identified a single residue, Tyr[100], as the main mediator of sugar discrimination in human PrimPol. Structural analyses and sequence comparison with other members of the AEP superfamily enabled us to predict and demonstrate that a change of Tyr[100] to histidine frees PrimPol to efficiently incorporate NTPs during polymerase and primase reactions in vitro, and provides increased resistance to downregulation of dNTP pools during DNA replication in murine embryonic fibroblasts (MEFs) and U2OS osteosarcoma cells. The very same mutation in PrimPol (Y100H) has been identified in lung cancer[30], suggesting its implication to promote survival during the tumorigenesis process

Methods

Results

Conclusion