Homologous Recombination Is Stimulated by a Decrease in dUTPase in Arabidopsis

Emeline Dubois,Séverine Domenichini,Nicolas Siaud,Bertrand Gakière,Florence Guérard,Sophie Massot,Teresa Roldan-Arjona,Dolores Córdoba-Cañero,Marie-Pascale Doutriaux,Michael Lichten

doi:10.1371/journal.pone.0018658

Abstract

Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants.

Highlights

Deamination of cytosine (C) results in the formation of uracil (U) in DNA which will code for adenine in the following replication cycle, giving rise to a C to T transition [1,2]
The deoxyuridine triphosphatase (dUTPase) performs two functions: keeping a low dUTP level in the cell and allowing dTTP synthesis. 5-fluorouracil (5FU) inhibits the thymidilate synthase decreasing dTTP synthesis, which leads to an increase in the dUTP/dTTP ratio which becomes more severe when the dUTPase is depleted [5]. 5-FU can incorporate into DNA, adding to the damage due to dTTP depletion, via its subsequent excision
Wild-type (WT) E. coli transformed with an empty vector remained sensitive to the presence of uracil or 5FU, whereas the growth of this same dut-1 mutant on uracil or 5FU containing medium was restored to a similar level as the WT E. coli upon transformation with the AtDUT1 cDNA (Figure 1A)

Summary

Introduction

Deamination of cytosine (C) results in the formation of uracil (U) in DNA which will code for adenine in the following replication cycle, giving rise to a C to T (thymine) transition [1,2]. DNA polymerase can incorporate dUTP (deoxyuridine triphosphate) instead of dTTP (deoxythymidine triphosphate) [3]. To prevent such a misincorporation, a low dUTP to dTTP ratio must be maintained [4]. DUMP can be used as a substrate by thymidylate synthase to initiate the synthesis of dTTP. Through this reaction, the dUTPase performs two functions: keeping a low dUTP level in the cell and allowing dTTP synthesis. Uracils in DNA are repaired by the base excision repair (BER) pathway. During the BER process, a single-strand break can be transformed into a double-strand break (DSB) if replication occurs at a nicked DNA site [7]

Methods

Results

Conclusion