UNG-1 and APN-1 are the major enzymes to efficiently repair 5-hydroxymethyluracil DNA lesions in C. elegans

Arturo Papaluca,H Uri Saragovi,Dindial Ramotar,J Richard Wagner

doi:10.1038/s41598-018-25124-1

Abstract

In Caenorhabditis elegans, two DNA glycosylases, UNG-1 and NTH-1, and two AP endonucleases, APN-1 and EXO-3, have been characterized from the base-excision repair (BER) pathway that repairs oxidatively modified DNA bases. UNG-1 removes uracil, while NTH-1 can remove 5-hydroxymethyluracil (5-hmU), an oxidation product of thymine, as well as other lesions. Both APN-1 and EXO-3 can incise AP sites and remove 3′-blocking lesions at DNA single strand breaks, and only APN-1 possesses 3′- to 5′-exonulease and nucleotide incision repair activities. We used C. elegans mutants to study the role of the BER pathway in processing 5-hmU. We observe that ung-1 mutants exhibited a decrease in brood size and lifespan, and an elevated level of germ cell apoptosis when challenged with 5-hmU. These phenotypes were exacerbated by RNAi downregulation of apn-1 in the ung-1 mutant. The nth-1 or exo-3 mutants displayed wild type phenotypes towards 5-hmU. We show that partially purified UNG-1 can act on 5-hmU lesion in vitro. We propose that UNG-1 removes 5-hmU incorporated into the genome and the resulting AP site is cleaved by APN-1 or EXO-3. In the absence of UNG-1, the 5-hmU is removed by NTH-1 creating a genotoxic 3′-blocking lesion that requires the action of APN-1.

Highlights

Endogenous and exogenous reactive oxygen species (ROS), such as superoxide radical anions and hydrogen peroxide generate hydroxyl radicals that react with DNA to induce a variety of DNA damage[1]
We report the surprising finding that ung[1], and not nth-1, mutants showed a number of phenotypes that are associated with a defect in DNA damage response when the animals were challenged with 5-hmU, suggesting that UNG-1 is the major DNA glycosylase involved in processing 5-hmU lesions
C. elegans mutants deficient in both APN-1 and EXO-3 are hypersensitive to DOX, methyl methanesulfonate (MMS)- and CDDP-induced DNA lesions

Summary

Introduction

Endogenous and exogenous reactive oxygen species (ROS), such as superoxide radical anions and hydrogen peroxide generate hydroxyl radicals that react with DNA to induce a variety of DNA damage[1]. Unlike the apn-1(tm6691) mutant, the exo-3(tm4374) mutants treated with either 5-hmU or DOX, respectively, showed an average of 2.7 ± 1.1 or 5.1 ± 1.2 apoptotic cells per animal, which was comparable to the wild type (Fig. 3B[vii and xi vs v and ix] and D,E), suggesting that EXO-3 has minimal role compared to APN-1 in processing 5-hmU and DOX-induced DNA lesions.

Results

Conclusion