Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1

Background

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases.

Methods

Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4^cat and UNG from different structural superfamilies were used.

Results

We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1.

Conclusions

We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4^cat, AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions.

General significance

Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.     

Genome and cancer single nucleotide polymorphisms of the human NEIL1 DNA glycosylase: Activity,structure, and the effect of editing

The repair of free-radical oxidative DNA damage is carried out by lesion-specific DNA glycosylases as the first step of the highly conserved base excision repair (BER) pathway. In humans, three orthologs of the prototypical endonuclease VIII (Nei), the Nei-like NEIL1-3 enzymes are involved in the repair of oxidized DNA lesions. In recent years, several genome and cancer single-nucleotide polymorphic variants of the NEIL1 glycosylase have been identified. In this study we characterized four variants of human NEIL1: S82C, G83D, P208S, and ΔE28, and tested their ability to excise pyrimidine-derived lesions such as thymine glycol (Tg), 5-hydroxyuracil (5-OHU), and dihydrouracil (DHU) and the purine-derived guanidinohydantoin (Gh), spiroiminodihydantoin 1 (Sp1), and methylated 2,6-diamino-4-hydroxy-5-formamidopyrimidine (MeFapyG). The P208S variant has near wild-type activity on all substrates tested. The S82C and ΔE28 variants exhibit decreased Tg excision compared to wild-type. G83D displays little to no activity with any of the substrates tested, with the exception of Gh and Sp1. Human NEIL1 is known to undergo editing whereby the lysine at position 242 is recoded into an arginine. The non-edited form of NEIL1 is more efficient at cleaving Tg than the R242 form, but the G83D variant does not cleave Tg regardless of the edited status of NEIL1. The corresponding G86D variant in Mimivirus Nei1 similarly lacks glycosylase activity. A structure of a G86D–DNA complex reveals a rearrangement in the β4/5 loop comprising Leu84, the highly-conserved void-filling residue, thereby providing a structural rationale for the decreased glycosylase activity of the glycine to aspartate variant.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.     

Biochemical mapping of human NEIL1 DNA glycosylase and AP lyase activities

Base excision repair of oxidized DNA in human cells is initiated by several DNA glycosylases with overlapping substrate specificity. The human endonuclease VIII homologue NEIL1 removes a broad spectrum of oxidized pyrimidine and purine lesions. In this study of NEIL1 we have identified several key residues, located in three loops lining the DNA binding cavity, important for lesion recognition and DNA glycosylase/AP lyase activity for oxidized bases in double-stranded and single-stranded DNA. Single-turnover kinetics of NEIL1 revealed that removal of 5-hydroxycytosine (5-OHC) and 5-hydroxyuracil (5-OHU) is ～25 and ～10-fold faster in duplex DNA compared to single-stranded DNA, respectively, and also faster than removal of dihydrothymine (DHT) and dihydrouracil (DHU), both in double-stranded and single-stranded DNA. NEIL1 excised 8-oxoguanine (8-oxoG) only from double-stranded DNA and analysis of site-specific mutants revealed that Met81, Arg119 and Phe120 are essential for removal of 8-oxoG. Further, several arginine and histidine residues located in the loop connecting the two β-strands forming the zincless finger motif and projecting into the DNA major groove, were shown to be imperative for lesion processing for both single- and double-stranded substrates. Trapping experiments of active site mutants revealed that the N-terminal Pro2 and Lys54 can alternate to form a Schiff-base complex between the protein and DNA. Hence, both Pro2 and Lys54 are involved in the AP lyase activity. While wildtype NEIL1 activity almost exclusively generated a δ-elimination product when processing single-stranded substrates, substitution of Lys54 changed this in favor of a β-elimination product. These results suggest that Pro2 and Lys54 are both essential for the concerted action of the β,δ-elimination in NEIL1.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3′ and 5′ ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as described previously. On the basis of ¹⁸O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair.     

Structural characterization of viral ortholog of human DNA glycosylase NEIL1 bound to thymine glycol or 5-hydroxyuracil-containing DNA

Thymine glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublié, S. (2009) J. Biol. Chem. 284, 26174-26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site.     

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.     

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

Interstrand DNA–DNA cross-links are highly toxic lesions that are important in medicinal chemistry, toxicology, and endogenous biology. In current models of replication-dependent repair, stalling of a replication fork activates the Fanconi anemia pathway and cross-links are “unhooked” by the action of structure-specific endonucleases such as XPF-ERCC1 that make incisions flanking the cross-link. This process generates a double-strand break, which must be subsequently repaired by homologous recombination. Recent work provided evidence for a new, incision-independent unhooking mechanism involving intrusion of a base excision repair (BER) enzyme, NEIL3, into the world of cross-link repair. The evidence suggests that the glycosylase action of NEIL3 unhooks interstrand cross-links derived from an abasic site or the psoralen derivative trioxsalen. If the incision-independent NEIL3 pathway is blocked, repair reverts to the incision-dependent route. In light of the new model invoking participation of NEIL3 in cross-link repair, we consider the possibility that various BER glycosylases or other DNA-processing enzymes might participate in the unhooking of chemically diverse interstrand DNA cross-links.     

Expression and purification of NEIL3, a human DNA glycosylase homolog

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.     

NEIL1 is the major DNA glycosylase that processes 5-hydroxyuracil in the proximity of a DNA single-strand break

5-Hydroxyuracil (5-OHU) in DNA, arising during endogenous DNA damage and caused by ionizing radiation, is removed by the base excision repair pathway. However, in addition to base lesions, ionizing radiation also generates DNA single-strand breaks (SSBs). When these DNA lesions are located in the proximity of each other, this may result in a profound effect on both repair of the damaged base and the SSB. We therefore examined the repair of DNA substrates containing 5-OHU lesions in the proximity of the 3'-end of a SSB. We found that SSB repair by DNA ligase IIIalpha and DNA polymerase beta is impaired by the presence of the nearby 5-OHU lesion, indicating the requirement for a DNA glycosylase which would be able to remove 5-OHU before SSB repair. Subsequently, we found that although both SMUG1 and NEIL1 are able to excise 5-OHU lesions located in the proximity of the 3'-end of a DNA SSB, NEIL1 is more efficient in the repair of these DNA lesions.     

AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair

Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3′–5′ exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3′–5′ exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER.     

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.     

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K_d = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.     

Structural and functional properties of CiNTH, an endonuclease III homologue of the ascidian Ciona intestinalis: critical role of N-terminal region

Oxidatively damaged bases in DNA can cause cell death, mutation and/or cancer induction. To overcome such deleterious effects of DNA base oxidation, cells are equipped with base excision repair (BER) initiated by DNA glycosylases. Endonuclease III (Nth), a major DNA glycosylase, mainly excises oxidatively damaged pyrimidines from DNA. The aims of this study were to obtain an overview of the repair mechanism of oxidatively damaged bases and to elucidate the function of BER in maintaining genome stability during embryogenesis and development. In this study, we used the ascidian Ciona intestinalis because at every developmental stage it is possible to observe the phenotype of individuals with DNA damage or mutations. Sequence alignment analysis revealed that the amino acid sequence of Ciona intestinalis Nth homologue (CiNTH) had high homology with those of Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and human Nth homologues. It was evident that two domains, the Helix-hairpin-Helix and 4Fe-4S cluster domains that are critical regions for the Nth activity, are well conserved in CiNTH. CiNTH efficiently complemented the sensitivity of E. coli nth nei mutant to H(2)O(2). CiNTH was bifunctional, with DNA glycosylase and AP lyase activities. It removed thymine glycol, 5-formyluracil and 8-oxoguanine paired with G from DNA via a β-elimination reaction. Interestingly, the N-terminal 44 amino acids were essential for the DNA glycosylase activity of CiNTH.     

Processing of N5-substituted formamidopyrimidine DNA adducts by DNA glycosylases NEIL1 and NEIL3

A variety of agents cause DNA base alkylation damage, including the known hepatocarcinogen aflatoxin B₁ (AFB₁) and chemotherapeutic drugs derived from nitrogen mustard (NM). The N7 site of guanine is the primary site of alkylation, with some N7-deoxyguanosine adducts undergoing imidazole ring-opening to stable mutagenic N⁵-alkyl formamidopyrimidine (Fapy-dG) adducts. These adducts exist as a mixture of canonical β- and unnatural α-anomeric forms. The β species are predominant in double-stranded (ds) DNA. Recently, we have demonstrated that the DNA glycosylase NEIL1 can initiate repair of AFB₁-Fapy-dG adducts both in vitro and in vivo, with Neil1^−/− mice showing an increased susceptibility to AFB₁-induced hepatocellular carcinoma.Here, we hypothesized that NEIL1 could excise NM-Fapy-dG and that NEIL3, a closely related DNA glycosylase, could excise both NM-Fapy-dG and AFB₁-Fapy-dG. Product formation from the reaction of human NEIL1 with ds oligodeoxynucleotides containing a unique NM-Fapy-dG followed a bi-component exponential function under single turnover conditions. Thus, two adduct conformations were differentially recognized by hNEIL1. The excision rate of the major form (∼13.0 min⁻¹), presumed to be the β-anomer, was significantly higher than that previously reported for 5-hydroxycytosine, 5-hydroxyuracil, thymine glycol (Tg), and AFB₁-Fapy-dG. Product generation from the minor form was much slower (∼0.4 min⁻¹), likely reflecting the rate of conversion of the α anomer into the β anomer. Mus musculus NEIL3 (MmuNEIL3Δ324) excised NM-Fapy-dG from single-stranded (ss) DNA (turnover rate of ∼0.4 min⁻¹), but not from ds DNA. Product formation from ss substrate was incomplete, presumably because of a substantial presence of the α anomer. MmuNEIL3Δ324 could not initiate repair of AFB₁-Fapy-dG in either ds or ss DNA. Overall, the data suggest that both NEIL1 and NEIL3 may protect cells against cytotoxic and mutagenic effects of NM-Fapy-dG, but NEIL1 may have a unique role in initiation of base excision repair of AFB₁-Fapy-dG.     

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.     

Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the base excision repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of Escherichia coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity with the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5'2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the "zincless finger" motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNei1. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates.