Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Dolores Córdoba-Cañero,Emeline Dubois,Rafael R Ariza,Marie-Pascale Doutriaux,Teresa Roldán-Arjona

doi:10.1074/jbc.m109.067173

Abstract

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung(-/-) mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung(-/-) mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.

Highlights

Cells minimize dUTP misincorporation by maintaining a low dUTP/dTTP ratio through the enzyme deoxyuridine triphosphate nucleotidohydrolase, which catalyzes the hydrolysis of dUTP to dUMP [5]
Uracil arising in DNA either from misincorporation of dUMP or from deamination of cytosine is actively removed through the multistep base excision repair (BER)3 pathway [6]
AtUNG Encodes a Family-1 Uracil DNA Glycosylase—The Arabidopsis genome contains two putative genes (At3g18630 and At2g10550) encoding polypeptides with sequence similarity to uracil DNA glycosylases

Summary

Introduction

Cells minimize dUTP misincorporation by maintaining a low dUTP/dTTP ratio through the enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase), which catalyzes the hydrolysis of dUTP to dUMP [5]. Uracil arising in DNA either from misincorporation of dUMP or from deamination of cytosine is actively removed through the multistep base excision repair (BER) pathway [6]. Family-1 enzymes, such as human UNG, recognize uracil in an extrahelical conformation and are active both on dsDNA and ssDNA. Family-3 UDGs are represented by SMUG1 from vertebrates, are active both on dsDNA and ssDNA, and have a broader specificity than family-1 enzymes. In addition to the five UDG families, at least three families of the HhH-GPD superfamily of DNA glycosylases (named after its hallmark helix-hairpin-helix and Gly/Pro-rich loop followed by a conserved aspartate) include members with UDG activity [6, 9]. We report that AtUNG, a member of the Family-1 UDGs, is the major UDG activity in Arabidopsis cell extracts and is absolutely required for initiation of BER of uracil in vitro.

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Conclusion