Differential control of dNTP biosynthesis and genome integrity maintenance by the dUTPase superfamily enzymes

Rita Hirmondo,Judit Toth,Beata G Vertessy,Eva Viola Suranyi,Anna Lopata

doi:10.1038/s41598-017-06206-y

Rita Hirmondo, Judit Toth + Show 3 more

Open Access

https://doi.org/10.1038/s41598-017-06206-y

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Abstract

dUTPase superfamily enzymes generate dUMP, the obligate precursor for de novo dTTP biosynthesis, from either dUTP (monofunctional dUTPase, Dut) or dCTP (bifunctional dCTP deaminase/dUTPase, Dcd:dut). In addition, the elimination of dUTP by these enzymes prevents harmful uracil incorporation into DNA. These two beneficial outcomes have been thought to be related. Here we determined the relationship between dTTP biosynthesis (dTTP/dCTP balance) and the prevention of DNA uracilation in a mycobacterial model that encodes both the Dut and Dcd:dut enzymes, and has no other ways to produce dUMP. We show that, in dut mutant mycobacteria, the dTTP/dCTP balance remained unchanged, but the uracil content of DNA increased in parallel with the in vitro activity-loss of Dut accompanied with a considerable increase in the mutation rate. Conversely, dcd:dut inactivation resulted in perturbed dTTP/dCTP balance and two-fold increased mutation rate, but did not increase the uracil content of DNA. Thus, unexpectedly, the regulation of dNTP balance and the prevention of DNA uracilation are decoupled and separately brought about by the Dcd:dut and Dut enzymes, respectively. Available evidence suggests that the discovered functional separation is conserved in humans and other organisms.

Highlights

Proper control of the intracellular concentration of deoxyribonucleoside-5-triphosphates, the building blocks of DNA, is critically important for efficient and high-fidelity DNA replication and genomic stability[1, 2]
Dut catalyzes the break-down of dUTP to dUMP and with this action it potentially takes part i) in dTTP biosynthesis, ii) in the maintenance of low dUTP/dTTP ratio to prevent uracil incorporation into DNA
This mutant presents an extremely low catalytic activity (Fig. 3A, Table 1) to what was observed in the E. coli enzyme[24]

Summary

Introduction

Proper control of the intracellular concentration of deoxyribonucleoside-5-triphosphates (dNTPs), the building blocks of DNA, is critically important for efficient and high-fidelity DNA replication and genomic stability[1, 2]. The enzymes that catalyze these conversions belong to the dUTPase superfamily comprising dCTP deaminase (Dcd), dUTPase (Dut) and the bifunctional dCTP deaminase/dUTPase (Dcd:dut) (Fig. 1). In addition to dUMP production, the dUTPase reaction serves to eliminate excess dUTP to prevent uracil incorporation into DNA in place of thymine[7, 8]. In high dUTP/dTTP ratios, DNA polymerases keep re-incorporating dUTP and the repair process becomes overwhelmed. Dut catalyzes the break-down of dUTP to dUMP and with this action it potentially takes part i) in dTTP biosynthesis, ii) in the maintenance of low dUTP/dTTP ratio to prevent uracil incorporation into DNA www.nature.com/scientificreports/. As dTTP biosynthesis is an essential process and a major target in several current drug therapies, it is important to pinpoint those pathways in which Dut is a key contributing enzyme

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