Abstract
Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (beta-delta) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression.
Highlights
A potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth, or its mammalian homologue NTH1
We report that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei
The mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression
Summary
8-oxoG, 8-oxoguanine; AP, apurinic/ apyrimidinic; Tg, thymine glycol; BER, base excision repair; 5-OHU, 5-hydroxyuracil; Ni-NTA, nickel-nitrilotriacetic acid; HPLC, high pressure liquid chromatography; H2TH, helix-two-turn-helix; TCR, transcription-coupled repair. Nth is one of the most widespread DNA glycosylases in eukaryotes, as well as prokaryotes, whereas the Nei orthologs are rarely found in bacteria and are even absent in the Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Drosophila melanogaster genomes. This distribution suggests that mammalian species may lack Nei homologues, three Fpg/Nei DNA glycosylase-like sequences have recently been registered in a full-length cDNA database Because we found three human NEIL genes in the database, we analyzed the enzymatic activity of the proteins in vitro and in vivo and their relationships to residual glycosylase activities in mNth knock-out mice
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