Elsevier

DNA Repair

Volume 1, Issue 7, 17 July 2002, Pages 517-529
DNA Repair

A novel human DNA glycosylase that removes oxidative DNA damage and is homologous to Escherichia coli endonuclease VIII

https://doi.org/10.1016/S1568-7864(02)00036-8Get rights and content

Abstract

Prokaryotes and lower eukaryotes possess redundant activities that remove the plethora of oxidative DNA base damages produced during normal oxidative metabolism and which have been associated with cancer and aging. Thus far, only one oxidized pyrimidine-specific DNA glycosylase has been identified in humans, hNthl. Here, we report the identification of three new putative human DNA glycosylases that are phylogenetically members of the Fpg/Nei family primarily found in the bacterial kingdom. We have characterized one of these, hNEI1, and show it to be functionally homologous to bacterial Nei, that is, its principal substrates are oxidized pyrimidines, it undergoes a lyase reaction by, β,δ-elimination and traps a Schiff base with a substrate containing thymine glycol (Tg). Furthermore, inactivation of active site residues shown to be important in Escherichia coli Nei inactivate the human enzyme. The hNEI1 gene is located on the long arm of chromosome 15 that is frequently deleted in human cancers.

Introduction

Highly reactive hydroxyl radicals, formed as a byproduct of oxidative metabolism, produce a broad spectrum of oxidized DNA bases including potentially mutagenic and cytotoxic pyrimidine lesions (for a review see [1]). Such oxidative DNA lesions have been implicated in cancer, neurodegenerative diseases and aging. Oxidized DNA bases are recognized and removed from DNA by a class of enzymes called DNA glycosylases, which initiate the first step in base excision repair by cleaving the damaged base and introducing a strand break in the DNA via its associated lyase reaction (for reviews see [1], [2], [3]). The DNA glycosylases that recognize and remove oxidatively damaged DNA bases are highly similar in sequence and are distributed across all three kingdoms. In prokaryotes and lower eukaryotes, which are amenable to genetic analysis, cells completely devoid of the DNA glycosylases that recognize oxidized DNA bases exhibit a high spontaneous mutation frequency [4], [5], [6], [7]. Escherichia coli cells lacking oxidized pyrimidine-specific DNA glycosylases are also hypersensitive to cytotoxic agents [6] and spontaneous mutations are targeted to oxidized cytosine residues [8] which are readily bypassed and mispair [9], [10]. The most common oxidative DNA glycosylase with orthologs in all three kingdoms is endonuclease III (EcNth), originally identified and characterized in the bacterium E. coli and encoded by the Nth gene (for reviews see [1], [2], [3]. The principal substrates for EcNth and its orthologs are oxidized pyrimidines and formamidopyrimidines [1], [2], [3], [11]. Eukaryotic orthologs of Nth have been cloned and the human activity, hNTH1, closely resembles that of the E. coli and Saccharomyces cerevisiae orthologs [11], [12], [13]. To date hNTH1 is the only human DNA glycosylase demonstrated to remove oxidized pyrimidines. Since redundant activities are present in prokaryotes and lower eukaryotes it seemed likely that a redundant activity(ies) would be found in vertebrates. A reasonable target for this role is a human homolog of endonuclease VIII (EcNei) which like hNTH1 removes oxidized pyrimidines [14]. EcNei is a member of the Fpg/Nei family of DNA glycosylases. The other member of the Fpg/Nei family, formamidopyrimidine DNA glycosylase or MutM (EcFpg), removes 7,8-dihydro-8-oxoguanine (8-oxoG) paired with C [5]. Although EcFpg and EcNei have different substrate specificities, they share common N- and C-terminal domains and a similar mode of action [15]. Fpg is primarily distributed in the bacterial kingdom, although an Fpg ortholog has been characterized in Arabidopsis thaliana (AtFpg) [16], [17]. Putative orthologs of EcNei have been identified in only a few bacterial species.

Here, we report the existence of orthologs of Fpg/Nei family members in mouse and humans, as well as in other eukaryotes; we have identified three putative homologs of EcNei in humans. The human NEI proteins share the characteristic motifs of the Fpg/Nei family as well as most of the key residues shown to be involved in catalysis in the prokaryotic Fpg/Nei proteins. Phylogenetic analysis suggests that Nei may have arisen in metazoans or in Gram positive bacteria, but came to the proteobacteria via gene transfer. We also report the cloning, expression and characterization of a human NEI ortholog (hNEI1) and show its activity to be the same as that of E. coli Nei protein.

Section snippets

Identification of human NEI homologs

A BLAST search of the NCBI non-redundant protein database based on the AtFpg sequence identified three human proteins, designated hNEI1–hNEI3, with expectation values ranging from 0.0005 to 0.012. Additional eukaryotic Fpg/Nei homologs were identified in other metazoans including fish (EST), divergent plants, and a fungus by searching GenBank and dbEST. These results suggest that a number of different eukaryotes possess members of the Fpg/Nei family.

Fig. 1 shows the alignment of the three human

Concluding remarks

While this manuscript was under review, Hazra et al. [32] reported the cloning and characterization of hNEI1 and the identification of hNEI2. Although fewer pyrimidine substrates were used in the Hazra et al. studies, their characterization of substrate specificity basically agrees with our observations, that is, hNEI1 has a similar substrate specificity to EcNei insofar as oxidized pyrimidines are better substrates than 8-oxoG. Although the sequence reported for hNEI2 is congruent with our

Enzymes, oligonucleotides and other reagents

Full length cDNA clone, HRC08117 was obtained from Dr. Sumio Sugano (Human Genome Center, University of Tokyo, Japan). The pET system (Novagen) or IMPACT CN system (New England Biolabs) were employed to purify recombinant E. coli (Fpg, endonucleases III, IV and VIII) and human (hNTH1 and hOGG1) DNA glycosylases using our laboratory procedures (unpublished data). Oligonucleotides containing oxidative DNA damages (DHT, DHU and 8-oxoG) were purchased from Operon technologies. Oligonucleotides

Acknowledgements

This work was supported by NIH PHS R37 CA33657 awarded to S.S.W. by the National Cancer Institute. The authors are grateful to Dr. Sumio Sugano (University of Tokyo, Japan) for sending us the full length cDNA clone containing hNEI1. The computational studies were supported by the Molecular Modeling Facility of the Vermont Cancer Center.

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