Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA

Abstract

8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase͞AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase͞AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replicationassociated repair of oxidized bases.

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.…”

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

“…The human bi-functional glycosylases NEIL1 and NEIL2 process oxidized base damage in a fashion similar to OGG1 and NTH1, however NEIL1/2 further processes the 3'-unsaturated aldehyde through a ß,δ-elimination mechanism to yield a 3'phosphate and 5'phosphate at the margins (Table I) [46]. While APE1 can efficiently process the ß-elimination product of OGG1 and NTH1, its DNA 3'-phosphatase activity needed for the processing of the ß,δ-elimination product produced by NEIL1/2 is extremely weak [24].…”

“…As described above, NEIL glycosylase substrates undergo ß,δ-elimination to yield phosphate groups on both the 3' and 5' ends. PNKP efficiently removes this replication block generating a 3'OH moiety [46]. Furthermore, a stable complex of NEIL2, PNKP, XRCC1, LigIIIα, and pol ß was isolated as a 'repairosome' from human cells [23].…”