Human DNA glycosylases of the bacterial Fpg/MutM superfamily: an alternative pathway for the repair of 8-oxoguanine and other oxidation products in DNA

Abstract

The mild phenotype associated with targeted disruption of the mouse OGG1 and NTH1 genes has been attributed to the existence of back-up activities and/or alternative pathways for the removal of oxidised DNA bases. We have characterised two new genes in human cells that encode DNA glycosylases, homologous to the bacterial Fpg (MutM)/Nei class of enzymes, capable of removing lesions that are substrates for both hOGG1 and hNTH1. One gene, designated HFPG1, showed ubiquitous expression in all tissues examined whereas the second gene, HFPG2, was only expressed at detectable levels in the thymus and testis. Transient transfections of HeLa cells with fusions of the cDNAs to EGFP revealed intracellular sorting to the nucleus with accumulation in the nucleoli for hFPG1, while hFPG2 co-localised with the 30 kDa subunit of RPA. hFPG1 was purified and shown to act on DNA substrates containing 8-oxoguanine, 5-hydroxycytosine and abasic sites. Removal of 8-oxoguanine, but not cleavage at abasic sites, was opposite base-dependent, with 8-oxoG:C being the preferred substrate and negligible activity towards 8-oxoG:A. It thus appears that hFPG1 has properties similar to mammalian OGG1 in preventing mutations arising from misincorporation of A across 8-oxoG and could function as a back-up repair activity for OGG1 in ogg1(-/-) mice.

Figure 1

(A) Aligned sequences of hFPG1 (accession no. BAB15337), hFPG2 (NP_060718), E.coli Fpg (FPG_ECOLI) and E.coli Nei (END8_ECOLI). The conserved N-terminal domain and the helix–two-turns–helix motif are boxed in red. The conserved zinc finger motifs of E.coli Fpg, Nei and hFPG2 are boxed in green. Putative nuclear localisation signals of hFPG1 and hFPG2 are boxed in blue. The RANbp-like zinc finger motif and the duplicated zinc ribbon domains of hFPG2 are boxed in purple. The alignment was made using T-Coffee v.1.37 (36) followed by manual editing and layout using GeneDoc (K.B.Nicholas and H.B.Nicholas, GeneDoc: a tool for editing and annotating multiple sequence alignments, distributed by the authors). (B) Alignment of the duplicated zinc ribbon domain of hFPG2 with those of related sequences in the protein database.

Figure 2

Northern blot analysis of HFPG1 and HFPG2 expression. Poly(A)⁺ mRNA (2 µg/lane) extracted from various normal human tissues as indicated were hybridised with full-length cDNAs of hFPG1 and hFPG2.

Figure 3

Nuclear localisation of the hFPG1–EGFP and hFPG2–EGFP fusion proteins. Exponentially growing asynchronous HeLa S3 cells were transiently transfected with constructs expressing hFPG1–EGFP (A–C), EGFP–hFPG2 (D–F and J–L) and pEGFP–N1 (G–I). The cells were imaged directly by fluorescence microscopy for EGFP detection (green, A, D, G and J). Nucleolin (red, B, E and H) and RPA (red, K) were both visualised by immunofluorescence with specific monoclonal antibodies followed by Alexa-conjugated secondary antibodies. DNA was stained by DAPI (blue, C, F, I and L).

Figure 4

FaPy DNA glycosylase activity of hFPG1 and hFPG2. (A) Different amounts of cell extracts from uninfected and baculovirus-infected insect cells expressing APE2, hFPG2 or hFPG1 from appropriate cDNA constructs were assayed for removal of faPy from [³H]-methyl-faPy-poly(dG·dC) (0.4 µg). Diamonds, hFPG1; small squares, hFPG2; triangles, APE2; large squares, uninfected. (B) Removal of faPy from [³H]-methyl-faPy-poly(dG·dC) DNA by increasing amounts of purified E.coli Fpg (triangles), hOGG1 (squares) and hFPG1 (diamonds).

Figure 5

8-OxoG DNA glycosylase activity of hFPG1. (A) An aliquot of 30 ng of purified E.coli Apn1, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 24 bp duplex oligodeoxyribonucleotide containing a single 8-oxoG residue opposite A, C, G or T. Strand cleavage was analysed by 20% PAGE and phosphorimaging. (B) Quantification of the strand cleavage reactions. Results represent the averages of three independent experiments and error bars indicate standard deviation.

Figure 6

5-ohC DNA glycosylase activity of hFPG1. (A) Incision and (B) probing for covalent hFPG1 DNA intermediates by NaCNBH₃ of 5-ohC-containing DNA by hFPG1. An aliquot of 30 ng of purified E.coli Nth, Nei, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 40 bp duplex oligodeoxyribonucleotide containing a single 5-ohC residue opposite G. Strand cleavage was analysed by 20% denaturing PAGE and bands detected by phosphorimaging. Protein–DNA complexes were separated by 10% Tricine–SDS–PAGE and detected by phosphorimaging. (C) Increasing amounts of purified E.coli Nei (diamonds), E.coli Fpg (triangles) and hFPG1 (squares) were incubated with 100 fmol of a 40 bp duplex oligodeoxyribonucleotide containing a single 5-ohC residue opposite G, and strand cleavage was quantified by 20% PAGE followed by phosphorimaging.

Figure 7

β,δ-elimination cleavage at abasic site DNA by hFPG1. (A) Incision and (B) probing for covalent hFPG1 DNA intermediates by NaCNBH₃ of AP DNA by hFPG1. An aliquot of 30 ng of purified E.coli Apn1, Nei, Nth, Fpg, hOGG1, hFPG1 or no enzyme was incubated with 100 fmol of a 25 bp duplex oligodeoxyribonucleotide containing a single abasic site opposite C. Strand cleavage was analysed by 20% PAGE and phosphorimaging. Protein–DNA complexes were separated by 10% Tricine–SDS–PAGE. (C) Opposite base-independent AP DNA cleaving activity of hFPG1. An aliquot of 30 ng of purified hOGG1 (white) or hFPG1 (grey) was incubated with 500 fmol of a 24 bp duplex oligodeoxyribonucleotide containing a single abasic site opposite A, C, G or T as indicated. Strand cleavage was quantified by 20% denaturing PAGE and phosphorimaging. The results shown represent the averages of three independent experiments.

Figure 8

Genomic structures of HFPG1 and HFPG2. (A) The regions around chromosomes 15q22.33 and (B) 4q34.2 are shown to scale. The sizes (in bp) of exons (striped boxes, untranslated exons; black boxes, translated exons) are shown above the line; the sizes of introns (in kb) are shown below the line. The positions of neighbouring genes are shown.