Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis.

Gustavo Carvalho,Alberto Díaz-Talavera,Patricia A. Calvo,Luis Blanco,María I. Martínez-Jiménez

doi:10.3390/genes12101487

Gustavo Carvalho, Alberto Díaz-Talavera + Show 3 more

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https://doi.org/10.3390/genes12101487

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Abstract

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

Highlights

Chain-terminating nucleotide analogues (CTNAs) are typically characterized by substitutions on the 30 C sugar pentose of nucleotides by elimination of the 30 -OH group
Mitochondrial Polγ was further classified as the most CTNA sensitive among the DNA polymerases found in human cells, because of its low discrimination between natural nucleotides and CTNAs [2]
The selectivity range of NRTIs on polymerases is, in general: HIV-reverse transcriptase >> Polγ > Polβ > Polαprimase = Polε; it could be inferred that mitochondria is a primary cellular target for CTNA inhibition by mainly affecting mitochondrial DNA synthesis, and DNA repair to a lesser extent, since Polβ localizes to the mitochondria [3,4]

Summary

Introduction

Chain-terminating nucleotide analogues (CTNAs) are typically characterized by substitutions on the 30 C sugar pentose of nucleotides by elimination of the 30 -OH group. When a DNA polymerase incorporates a CTNA that cannot be proofread, the DNA synthesis is interrupted due to the lack of the 30 -OH group required for phosphodiester bond formation with the incoming nucleotide. Nucleotide and nucleoside analogues have been widely and successfully used in antiviral therapy against HIV or hepatitis B as potent inhibitors of the viral reverse transcriptases (NRTIs). The selectivity range of NRTIs on polymerases is, in general: HIV-reverse transcriptase >> Polγ > Polβ > Polαprimase = Polε; it could be inferred that mitochondria is a primary cellular target for CTNA inhibition by mainly affecting mitochondrial DNA synthesis, and DNA repair to a lesser extent, since Polβ localizes to the mitochondria [3,4]. DdC has been claimed to be an opportunity to poison cancer cells that relies heavily on mitochondrial function impairing mtDNA replication [6]

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