Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.     

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k_obs of over 2500 min^?1.     

An evolutionary analysis of the helix-hairpin-helix superfamily of DNA repair glycosylases

The helix-hairpin-helix (HhH) superfamily of base excision repair DNA glycosylases is composed of multiple phylogenetically diverse enzymes that are capable of excising varying spectra of oxidatively and methyl-damaged bases. Although these DNA repair glycosylases have been widely studied through genetic, biochemical, and biophysical approaches, the evolutionary relationships of different HhH homologs and the extent to which they are conserved across phylogeny remain enigmatic. We provide an evolutionary framework for this pervasive and versatile superfamily of DNA glycosylases. Six HhH gene families (named AlkA: alkyladenine glycosylase; MpgII: N-methylpurine glycosylase II; MutY/Mig: A/G-specific adenine glycosylase/mismatch glycosylase; Nth: endonuclease III; OggI: 8-oxoguanine glycosylase I; and OggII: 8-oxoguanine glycosylase II) are identified through phylogenetic analysis of 234 homologs found in 94 genomes (16 archaea, 64 bacteria, and 14 eukaryotes). The number of homologs in each gene family varies from 117 in the Nth family (nearly every genome surveyed harbors at least one Nth homolog) to only five in the divergent OggII family (all from archaeal genomes). Sequences from all three domains of life are included in four of the six gene families, suggesting that the HhH superfamily diversified very early in evolution. The phylogeny provides evidence for multiple lineage-specific gene duplication events, most of which involve eukaryotic homologs in the Nth and AlkA gene families. We observe extensive variation in the number of HhH superfamily glycosylase genes present in different genomes, possibly reflecting major differences among species in the mechanisms and pathways by which damaged bases are repaired and/or disparities in the basic rates and spectra of mutation experienced by different genomes.     

Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.     

DNA glycosylase recognition and catalysis

DNA glycosylases are the enzymes responsible for recognizing base lesions in the genome and initiating base excision DNA repair. Recent structural and biochemical results have provided novel insights into DNA damage recognition and repair. The basis of the recognition of the oxidative lesion 8-oxoguanine by two structurally unrelated DNA glycosylases is now understood and has been revealed to involve surprisingly similar strategies. Work on MutM (Fpg) has produced structures representing three discrete reaction steps. The NMR structure of 3-methyladenine glycosylase I revealed its place among the structural families of DNA glycosylases and the X-ray structure of SMUG1 likewise confirmed that this protein is a member of the uracil DNA glycosylase superfamily. A novel disulfide cross-linking strategy was used to obtain the long-anticipated structure of MutY bound to DNA containing an A*oxoG mispair.     

Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates

Current methods to measure the fraction of active glycosylase molecules in a given enzyme preparation are slow and cumbersome. Here we report a novel assay for rapidly determining the active fraction based on molecular accessibility of a fluorescent DNA minor groove binder, 4′,6-diamidino-2-phenylindole (DAPI). Several 5,6-dihydrouracil-containing (DHU) DNA substrates were designed with sequence-dependent DAPI-binding sites to which base excision repair glycosylases were covalently trapped by reduction. Trapped complexes impeded the association of DAPI in a manner dependent on the enzyme used and the location of the DAPI-binding site in relation to the lesion. Of the sequences tested, one was shown to give an accurate measure of the fraction of active molecules for each enzyme tested from both the Fpg/Nei family and HhH-GPD Nth superfamily of DNA glycosylases. The validity of the approach was demonstrated by direct comparison with current gel-based methods. Additionally, the results are supported by in silico modeling based on available crystal structures.     

Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

Formamidopyrimidine-DNA glycosylase (Fpg; MutM) is a DNA repair enzyme widely distributed in bacteria. Fpg recognizes and excises oxidatively modified purines, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxoguanine (8-oxoG), with similar excision kinetics. It exhibits some lesser activity toward 8-oxoadenine. Fpg enzymes are also present in some plant and fungal species. The eukaryotic Fpg homologs exhibit little or no activity on DNA containing 8-oxoG, but they recognize and process its oxidation products, guanidinohydantoin (Gh) and spiroiminohydantoin (Sp). To date, several structures of bacterial Fpg enzymes unliganded or in complex with DNA containing a damaged base have been published but there is no structure of a eukaryotic Fpg. Here we describe the first crystal structure of a plant Fpg, Arabidopsis thaliana (AthFpg), unliganded and bound to DNA containing an abasic site analog, tetrahydrofuran (THF). Although AthFpg shares a common architecture with other Fpg glycosylases, it harbors a zincless finger, previously described in a subset of Nei enzymes, such as human NEIL1 and Mimivirus Nei1. Importantly the "αF-β9/10 loop" capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213-229 in Escherichia coli Fpg (EcoFpg) and corresponding to the "αF-β9/10 loop" does not affect the recognition and removal of oxidatively damaged DNA base lesions, with the exception of 8-oxoG. Although the exact role of the loop remains to be further explored, it is now clear that this protein segment is specific to the processing of 8-oxoG.     

Structural characterization of the Fpg family of DNA glycosylases

Until recently, the Fpg family was the only major group of DNA glycosylases for which no structural data existed. Prototypical members of this family, found in eukaryotes as well as prokaryotes, have now been crystallized as free proteins and as complexes with DNA. In this review, we analyze the available structural information for formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei). Special emphasis is placed on mechanisms by which these enzymes recognize and selectively excise cognate lesions from oxidatively damaged DNA. The problem of lesion recognition is considered in two parts: how the enzyme efficiently locates a single lesion embedded in a vast excess of DNA; and how the lesion is accommodated in a pocket near the active site of the enzyme. Although all crystal structures reported to date for the Fpg family lack the damaged base, functionally important residues that participate in DNA binding and enzyme catalysis have been clearly identified and other residues, responsible for substrate specificity, have been inferred.     

DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.     

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG–DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.     

Structure and activity of a thermostable thymine-DNA glycosylase: evidence for base twisting to remove mismatched normal DNA bases.

The repair of T:G mismatches in DNA is key for maintaining bacterial restriction/modification systems and gene silencing in higher eukaryotes. T:G mismatch repair can be initiated by a specific mismatch glycosylase (MIG) that is homologous to the helix-hairpin-helix (HhH) DNA repair enzymes. Here, we present a 2.0 A resolution crystal structure and complementary mutagenesis results for this thermophilic HhH MIG enzyme. The results suggest that MIG distorts the target thymine nucleotide by twisting the thymine base approximately 90 degrees away from its normal anti position within DNA. We propose that functionally significant differences exist in DNA repair enzyme extrahelical nucleotide binding and catalysis that are characteristic of whether the target base is damaged or is a normal base within a mispair. These results explain why pure HhH DNA glycosylases and combined glycosylase/AP lyases cannot be interconverted by simply altering their functional group chemistry, and how broad-specificity DNA glycosylase enzymes may weaken the glycosylic linkage to allow a variety of damaged DNA bases to be excised.     

Identification of a zinc finger domain in the human NEIL2 (Nei-like-2) protein

The recently identified human NEIL2 (Nei-like-2) protein, a DNA glycosylase/AP lyase specific for oxidatively damaged bases, shares structural features and reaction mechanism with the Escherichia coli DNA glycosylases, Nei and Fpg. Amino acid sequence analysis of NEIL2 suggested it to have a zinc finger-like Nei/Fpg. However, the Cys-X2-His-X16-Cys-X2-Cys (CHCC) motif present near the C terminus of NEIL2 is distinct from the zinc finger motifs of Nei/Fpg, which are of the C4 type. Here we show the presence of an equimolar amount of zinc in NEIL2 by inductively coupled plasma mass spectrometry. Individual mutations of Cys-291, His-295, Cys-315, and Cys-318, candidate residues for coordinating zinc, inactivated the enzyme by abolishing its DNA binding activity. H295A and C318S mutants were also shown to lack bound zinc, and a significant change in their secondary structure was revealed by CD spectra analysis. Molecular modeling revealed Arg-310 of NEIL2 to be a critical residue in its zinc binding pocket, which is highly conserved throughout the Fpg/Nei family. A R310Q mutation significantly reduced the activity of NEIL2. We thereby conclude that the zinc finger motif in NEIL2 is essential for its structural integrity and enzyme activity.     

Non-specific DNA binding interferes with the efficient excision of oxidative lesions from chromatin by the human DNA glycosylase,NEIL1

Although DNA in eukaryotes is packaged in nucleosomes, it remains vulnerable to oxidative damage that can result from normal cellular metabolism, ionizing radiation, and various chemical agents. Oxidatively damaged DNA is repaired in a stepwise fashion via the base excision repair (BER) pathway, which begins with the excision of damaged bases by DNA glycosylases. We reported recently that the human DNA glycosylase hNTH1 (human Endonuclease III), a member of the HhH GpG superfamily of glycosylases, can excise thymine glycol lesions from nucleosomes without requiring or inducing nucleosome disruption; optimally oriented lesions are excised with an efficiency approaching that seen for naked DNA [1]. To determine if this property is shared by human DNA glycoylases in the Fpg/Nei family, we investigated the activity of NEIL1 on defined nucleosome substrates. We report here that the cellular concentrations and apparent k_cat/K_M ratios for hNTH1 and NEIL1 are similar. Additionally, after adjustment for non-specific DNA binding, hNTH1 and NEIL1 proved to have similar intrinsic activities toward nucleosome substrates. However, NEIL1 and hNTH1 differ in that NEIL1 binds undamaged DNA far more avidly than hNTH1. As a result, hNTH1 is able to excise both accessible and sterically occluded lesions from nucleosomes at physiological concentrations, while the high non-specific DNA affinity of NEIL1 would likely hinder its ability to process sterically occluded lesions in cells. These results suggest that, in vivo, NEIL1 functions either at nucleosome-free regions (such as those near replication forks) or with cofactors that limit its non-specific binding to DNA.     

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

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, beta,delta-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.     

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A resolution. The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4). Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.     

New non-hydrolyzable substrate analogs for 8-oxoguanine-DNA glycosylases

8-Oxoguanine-DNA glycosylases play a key role in the repair of oxidatively damaged DNA. The Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are DNA base excision repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (oxoG) residue, and cleave DNA strand. Specific contacts between DNA phosphate groups and amino acids from active centers of these enzymes play a significant role in DNA-protein interactions. In order to design new non-hydrolyzable substrate analogs of Fpg and hOGG1 for structural studies modified DNA duplexes containing pyrophosphate or OEt-substituted pyrophosphate internucleotide (SPI) groups near the damage were tested. We showed that enzymes recognize and specifically bind to DNA duplexes obtained. The mechanism of incision of oxoG by the Fpg and hOGG1 was determined. We revealed that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the oxoG. In contrast, Fpg and hOGG1 effectively incise DNA duplex carrying analogous phosphate modifications 5' to the oxoG. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or substituted pyrophosphate groups immediately 3' to the oxoG are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a oxoG-containing DNA bound to catalytically active wild-type enzymes as well as their pro- and eucaryotic homologs.     

The novel DNA glycosylase,NEIL1, protects mammalian cells from radiation-mediated cell death

DNA damage mediated by reactive oxygen species generates miscoding and blocking lesions that may lead to mutations or cell death. Base excision repair (BER) constitutes a universal mechanism for removing oxidatively damaged bases and restoring the integrity of genomic DNA. In Escherichia coli, the DNA glycosylases Nei, Fpg, and Nth initiate BER of oxidative lesions; OGG1 and NTH1 proteins fulfill a similar function in mammalian cells. Three human genes, designated NEIL1, NEIL2 and NEIL3, encode proteins that contain sequence homologies to Nei and Fpg. We have cloned the corresponding mouse genes and have overexpressed and purified mNeil1, a DNA glycosylase that efficiently removes a wide spectrum of mutagenic and cytotoxic DNA lesions. These lesions include the two cis-thymineglycol(Tg) stereoisomers, guanine- and adenine-derived formamidopyrimidines, and 5,6-dihydrouracil. Two of these lesions, fapyA and 5S,6R thymine glycol, are not excised by mOgg1 or mNth1. We have also used RNA interference technology to establish embryonic stem cell lines deficient in Neil1 protein and showed them to be sensitive to low levels of gamma-irradiation. The results of these studies suggest that Neil1 is an essential component of base excision repair in mammalian cells; its presence may contribute to the redundant repair capacity observed in Ogg1 -/- and Nth1 -/- mice.     

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts

The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs.     

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases

DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix-hairpin-helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases.     

Repair of hydantoins, one electron oxidation product of 8-oxoguanine, by DNA glycosylases of Escherichia coli

8-Oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (k_cat/K_m of 0.24–0.26 min^–1 nM^–1), while Nei prefers G over C as the complementary base (k_cat/K_m – 0.15–0.17 min^–1 nM^–1). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG·A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp)·A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh·G or Sp·G pair, but not from Gh·C and Sp·C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.