Repair of oxidative DNA damage in Drosophila melanogaster: identification and characterization of dOgg1, a second DNA glycosylase activity for 8-hydroxyguanine and formamidopyrimidines

Abstract

In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using gamma-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from gamma-irradiated DNA. with k(ca)(t)/K:(M) values of 21.0 x 10(-5) and 11.2 x 10(-5) (min(-1) nM(-1)), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a ss-elimination reaction at the 3'-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.

Figure 1

Drosophila OGG1 sequence analysis. (A) Sequence alignment of the dOgg1 protein with its yeast (Sc), plant (At) and human (Hs) homologs. Black and gray boxes identify identical and similar residues respectively. Asterisks in the consensus line indicate conservation of the amino acid in all four proteins. (B) Structure of the dOGG1 gene. Boxes indicate exons.

Figure 2

Gel analysis of the dOgg1 purification steps. After expression in E.coli the purification fractions were analyzed by SDS–PAGE. M, molecular weight markers; lane 1, total cell lysate; lane 2, eluate from the glutathione–Sepharose 4B; lane 3, products of the thrombin cleavage reaction; lane 4, eluate from the MonoS column.

Figure 3

Excision of 8-OH-Gua and FapyGua by dOgg1 from DNA γ-irradiated under N₂O. Dark columns (1), pellets. Light columns (2), supernatant fractions. (Left) 100 µg of γ-irradiated DNA were incubated with 4 µg of heat inactivated dOgg1. (Right) 100 µg of γ-irradiated DNA were incubated with 4 µg of active dOgg1. The identification and the quantification of the products were achieved by GC/IDMS.

Figure 4

Cleavage activity of dOgg1 on DNA duplexes harboring single lesions. (A) Cleavage of 34mer oligodeoxyribonucleotides carrying a single 8-OH-Gua paired to each of the four normal DNA bases. (B) Cleavage of 34mer oligodeoxyribonucleotides carrying a single 8-OH-Ade paired to each of the four normal DNA bases. (C) Cleavage of 34mer oligodeoxyribonucleotides carrying a single AP site paired to each of the four normal DNA bases. The DNA duplexes were ³²P-labeled at the 5′-end of the strand carrying the lesion. Incubations were performed at 37°C for 15 min. The products were separated on 20% PAGE containing 7 M urea and quantified using a PhosphorImager.

Figure 5

Analysis of the products of the dOgg1 repair reaction. (A) Mechanism of strand cleavage. The 34mer carrying a single 8-OH-Gua residue was ³²P-labeled at its 5′ end and hybridized with a complementary sequence carrying a cytosine opposite the lesion. Incubation were performed with 10 ng of dOgg1, yOgg1 or Fpg protein for 15 min at 37°C. When indicated, reaction mixtures were then incubated with 10 ng of either endonuclease III (Nth) or endonuclease IV (Nfo). The products were separated by denaturing 20% PAGE. P1, P2 and P3 are the products described in the Results. (B) NaBH₄-mediated trapping assay. The 34mer carrying a single 8-OH-Gua residue was labeled at its 5′-end and hybridized with a complementary sequence carrying a cytosine opposite the lesion. Fifty nanograms of either Fpg, yOgg1 or dOgg1 proteins were allowed to react for 20 min at 37°C with the ³²P-labeled 34mer duplex DNA, containing a single 8-OH-Gua.C, in the presence of 50 mM of either NaCl or NaBH₄. The products of the reactions were separated using 15% SDS–PAGE.

Figure 6

Whole-mount in situ hybridization on stage 10B egg chambers (A and B) and third instar larvae imaginal discs (C and D). The antisense probe (A and C) reveals an expression of the dOGG1 transcript in germ cells in the egg chamber and a uniform expression in the imaginal disc, as the control sense probe (B and D) exhibits no staining.