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.

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

“…In contrast, the suppression of the mutations by 8-oxo-Gua by NTH1 and NEIL1 has come under scrutiny lately, due to their lack of activity and weak activity, respectively, for DNA duplexes containing 8-oxo-Gua:C [39, [41][42][43][44]. The findings reported herein provides the first evidence to demonstrate that NTH1 and NEIL1 suppress the mutagenesis induced by 8-oxo-Gua in living cells.…”

“…NEIL1 has the ability to excise not only 8-oxo-Gua in ds DNA [39,[41][42][43] but also 8-oxo-Gua in ss DNA [44]. In addition, proliferating cell nuclear antigen (PCNA) interacts with NEIL1 and stimulates NEIL1 activity with respect to excising 5-hydroxyuracil from ss DNA sequences, including fork structures [55].…”

“…In addition, the activity of NEIL1 for the 8-oxo-Gua:C pair, as well as 8-oxo-Gua in single-stranded (ss) DNA, is very low [39,[41][42][43][44]. However, these results may not reflect the in vivo situation, where its activity could be regulated by the presence of other proteins.…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…However, NEIL1 was later shown to prefer ring-opened purines, i.e., formamidopyrimidine (Fapy)-A and -G, which along with 8-oxoG are the preferred substrates for E. coli Fpg. While we initially showed 8-oxoG as a poor substrate of NEILs on duplex DNA substrate, others have shown NEIL1's robust 8-oxoG excision activity [54,56]. Subsequently, we showed that NEILs' activity and substrate specificity depend largely on the DNA structure, and NEILs have significant 5-hydroxyuracil excision activity towards single-stranded or bubble DNA [57].…”

“…Thus, both NTH1, preferring oxidized pyrimidines as substrates, and OGG1, primarily responsible for the removal of 8-oxoG and ring opened guanine, i.e., formamidopyrimidine (Fapy-G), catalyze β elimination [11,49]. Subsequently, we characterized two additional human DNA glycosylases (also independently discovered by others), which belong to the Fpg/Nei family; we named these NEIL1 and NEIL2 [50][51][52][53][54][55]. All these enzymes as already indicated are bifunctional glycosylases with broad substrate range.…”