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 gamma-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 2500min(-1).

Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA.

In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1-DNA product complex was disrupted by DNA polymerase β (POLβ) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLβ and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.

DNA 5-methylcytosine is a dynamic epigenetic mark with important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases, and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic-site processing during TET-TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG-substrate turnover. In early Xenopus embryos, Neil2 cooperates with Tdg in removing oxidized methylcytosines and specifying neural-crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP-site handover during oxidative DNA demethylation.

DNA methylation is an epigenetic mechanism that plays important roles in gene regulation and transposon silencing. Active DNA demethylation has evolved to counterbalance DNA methylation at many endogenous loci. Here, we report that active DNA demethylation also targets viral DNAs, tomato yellow leaf curl China virus (TYLCCNV) and its satellite tomato yellow leaf curl China betasatellite (TYLCCNB), to promote their virulence. We demonstrate that the βC1 protein, encoded by TYLCCNB, interacts with a ROS1-like DNA glycosylase in Nicotiana benthamiana and with the DEMETER (DME) DNA glycosylase in Arabidopsis thaliana. The interaction between βC1 and DME facilitates the DNA glycosylase activity to decrease viral DNA methylation and promote viral virulence. These findings reveal that active DNA demethylation can be regulated by a viral protein to subvert DNA methylation-mediated defense.

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.

GO system is part of base excision DNA repair and is required for the correct repair of 8-oxoguanine (8-oxoG), one of the most abundant oxidative lesions. Due to the ability of 8-oxoG to mispair with A, this base is highly mutagenic, and its repair requires two enzymes: Fpg that removes 8-oxoG from 8-oxoG:C pairs, and MutY that excises the normal A from 8-oxoG:A mispairs. Here we characterize the properties of putative GO system DNA glycosylases from Staphylococcus aureus, an important human opportunistic pathogen that causes hospital infections and presents a serious health concern due to quick spread of antibiotic-resistant strains. In addition to Fpg and MutY from the reference NCTC 8325 strain (SauFpg1 and SauMutY), we have also studied an Fpg homolog from a multidrug-resistant C0673 isolate (SauFpg2), which is different from SauFpg1 in its sequence. Both SauFpg enzymes showed the highest activity at pH 7.0-9.0 and NaCl concentrations 25-75 mM (SauFpg1) or 50-100 mM (SauFpg2), whereas SauMutY was active at a broad pH range and had a salt optimum at ∼75 mM NaCl. Both SauFpg1 and SauFpg2 bound and cleaved duplexes containing 8-oxoG, 5-hydroxyuracil, 5,6-dihydrouracil or apurinic/apyrimidinic site paired with C, T, or G, but not with A. For SauFpg1 and SauFpg2, 8-oxoG was the best substrate tested, and 5,6-dihydrouracil was the worst one. SauMutY efficiently excised adenine from duplex substrates containing A:8-oxoG or A:G pairs. SauFpg enzymes were readily trapped on DNA by NaBH4 treatment, indicating formation of a Schiff base reaction intermediate. Surprisingly, SauMutY was also trapped significantly better than its E. coli homolog. All three S. aureus GO glycosylases drastically reduced spontaneous mutagenesis when expressed in an fpg mutY E. coli double mutant. Overall, we conclude that S. aureus possesses an active GO system, which could possibly be targeted for sensitization of this pathogen to oxidative stress.

Following the formation of oxidatively-induced DNA damage, several DNA glycosylases are required to initiate repair of the base lesions that are formed. Recently, NEIL1 and other DNA glycosylases, including OGG1 and NTH1 were identified as potential targets in combination chemotherapeutic strategies. The potential therapeutic benefit for the inhibition of DNA glycosylases was validated by demonstrating synthetic lethality with drugs that are commonly used to limit DNA replication through dNTP pool depletion via inhibition of thymidylate synthetase and dihydrofolate reductase. Additionally, NEIL1-associated synthetic lethality has been achieved in combination with Fanconi anemia, group G. As a prelude to the development of strategies to exploit the potential benefits of DNA glycosylase inhibition, it was necessary to develop a reliable high-throughput screening protocol for this class of enzymes. Using NEIL1 as the proof-of-principle glycosylase, a fluorescence-based assay was developed that utilizes incision of site-specifically modified oligodeoxynucleotides to detect enzymatic activity. This assay was miniaturized to a 1536-well format and used to screen small molecule libraries for inhibitors of the combined glycosylase/AP lyase activities. Among the top hits of these screens were several purine analogs, whose postulated presence in the active site of NEIL1 was consistent with the paradigm of NEIL1 recognition and excision of damaged purines. Although a subset of these small molecules could inhibit other DNA glycosylases that excise oxidatively-induced DNA adducts, they could not inhibit a pyrimidine dimer-specific glycosylase.

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.

Although DNA methylation primarily represses transposable elements (TEs) in plants, it also represses select endosperm and pollen genes. These genes, or their cis-regulatory elements, are methylated in plant body tissues but are demethylated by DNA glycosylases (DNGs) in endosperm and pollen, enabling their transcription. Activity of either one of two DNGs, MDR1 or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen segregating mutations in both genes, we identified 58 candidate DNG target genes, whose expression is strongly decreased in double mutant pollen (124-fold decrease on average). These genes account for 11.1% of the wild-type pollen polyadenylated transcriptome, but they are silent or barely detectable in the plant body. They are unusual in their tendency to lack introns but even more so in their having TE-like methylation in their coding DNA sequence. Moreover, they are strongly enriched for predicted functions in cell wall modification. While some may support development of the pollen grain cell wall, expansins and pectinases in this set of genes suggest a function in cell wall loosening to support the rapid tip growth characteristic of pollen tubes as they carry the sperm cells through maternal apoplast and extracellular matrix of the pistil. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with potential for extremely high expression in pollen but constitutive silencing elsewhere.

High metabolic activity and low levels of antioxidant enzymes make neurons particularly prone to damage by reactive oxygen species. Thus, repair of oxidative DNA damage is essential for normal brain function. Base excision repair is the major pathway for repair of oxidative DNA damage, and is initiated by DNA glycosylases recognizing and removing the damaged base. In mammalian cells at least five different DNA glycosylases with overlapping substrate specificity, NEIL1, NEIL2, NEIL3, OGG1 and NTH1, remove oxidative DNA base lesions. Here we report mRNA expression and distribution of these five DNA glycosylases in human and rodent brains using in situ hybridization and Northern blotting supported by glycosylase activity assays. NEIL1, NEIL2, OGG1 and NTH1 showed widespread expression at all ages. In situ hybridization studies in mouse brain showed that expression of mNeil1 increased with age. In newborn mouse brain, mNeil3 revealed a discrete expression pattern in brain regions known to harbour stem cell populations, i.e., the subventricular zone, the rostral migratory stream, and the hilar region of the hippocampal formation. Expression of mNeil3 decreased with age, and in old mice brains could be detected only in layer V of neocortex. MNth1 was constitutively expressed during lifespan. In Northern blots, mOgg1 expression showed a transient decrease followed by an increase after 8 weeks of age. Assays for faPy DNA glycosylase activity revealed increased activity level with age in all brain regions analyzed. The widespread but differential expression of the DNA glycosylases recognizing oxidative base lesions suggests distinct and age dependent roles of these enzymes in genome maintenance in brain. The distribution of mNeil3 is particularly intriguing and points to a specific role of this enzyme in stem cell differentiation.

Unique DNA repair enzymes that provide self-resistance against therapeutically important, genotoxic natural products have been discovered in bacterial biosynthetic gene clusters (BGCs). Among these, the DNA glycosylase AlkZ is essential for azinomycin B production and belongs to the HTH_42 superfamily of uncharacterized proteins. Despite their widespread existence in antibiotic producers and pathogens, the roles of these proteins in production of other natural products are unknown. Here, we determine the evolutionary relationship and genomic distribution of all HTH_42 proteins from Streptomyces and use a resistance-based genome mining approach to identify homologs associated with known and uncharacterized BGCs. We find that AlkZ-like (AZL) proteins constitute one distinct HTH_42 subfamily and are highly enriched in BGCs and variable in sequence, suggesting each has evolved to protect against a specific secondary metabolite. As a validation of the approach, we show that the AZL protein, HedH4, associated with biosynthesis of the alkylating agent hedamycin, excises hedamycin-DNA adducts with exquisite specificity and provides resistance to the natural product in cells. We also identify a second, phylogenetically and functionally distinct subfamily whose proteins are never associated with BGCs, are highly conserved with respect to sequence and genomic neighborhood, and repair DNA lesions not associated with a particular natural product. This work delineates two related families of DNA repair enzymes-one specific for complex alkyl-DNA lesions and involved in self-resistance to antimicrobials and the other likely involved in protection against an array of genotoxins-and provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential. IMPORTANCE Bacteria are rich sources of secondary metabolites that include DNA-damaging genotoxins with antitumor/antibiotic properties. Although Streptomyces produce a diverse number of therapeutic genotoxins, efforts toward targeted discovery of biosynthetic gene clusters (BGCs) producing DNA-damaging agents is lacking. Moreover, work on toxin-resistance genes has lagged behind our understanding of those involved in natural product synthesis. Here, we identified over 70 uncharacterized BGCs producing potentially novel genotoxins through resistance-based genome mining using the azinomycin B-resistance DNA glycosylase AlkZ. We validate our analysis by characterizing the enzymatic activity and cellular resistance of one AlkZ ortholog in the BGC of hedamycin, a potent DNA alkylating agent. Moreover, we uncover a second, phylogenetically distinct family of proteins related to Escherichia coli YcaQ, a DNA glycosylase capable of unhooking interstrand DNA cross-links, which differs from the AlkZ-like family in sequence, genomic location, proximity to BGCs, and substrate specificity. This work defines two families of DNA glycosylase for specialized repair of complex genotoxic natural products and generalized repair of a broad range of alkyl-DNA adducts and provides a framework for targeted discovery of new compounds with therapeutic potential.

Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.

Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.

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 2.04Å 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.

Base excision repair (BER) is an evolutionarily conserved system which removes altered bases from DNA. The initial step in BER is carried out by DNA glycosylases which recognize altered bases and cut the N-glycosylic bond between the base and the DNA backbone. In kinetoplastid flagellates, such as Trypanosoma brucei, the modified base beta-D-glucosyl-hydroxymethyluracil (J) replaces a small percentage of thymine residues, predominantly in repetitive telomeric sequences. Base J is synthesized at the DNA level via the precursor 5-hydroxymethyluracil (5-HmU). We have investigated whether J in DNA can be recognized by DNA glycosylases from non-kinetoplastid origin, and whether the presence of J and 5-HmU in DNA has required modifications of the trypanosome BER system. We tested the ability of 15 different DNA glycosylases from various origins to excise J or 5-HmU paired to A from duplex oligonucleotides. No excision of J was found, but 5-HmU was excised by AlkA and Mug from Escherichia coli and by human SMUG1 and TDG, confirming previous reports. In a combination of database searches and biochemical assays we identified several DNA glycosylases in T. brucei, but in trypanosome extracts we detected no excision activity towards 5-HmU or ethenocytosine, a product of oxidative DNA damage and a substrate for Mug, TDG and SMUG1. Our results indicate that trypanosomes have a BER system similar to that of other organisms, but might be unable to excise certain forms of oxidatively damaged bases. The presence of J in DNA does not require a specific modification of the BER system, as this base is not recognized by any known DNA glycosylase.

The main purpose of this study was to determine whether enhancement of repair capacity would attenuate mitochondrial DNA oxidative damage and result in greater cell survival under stressful conditions. The repair of oxidative damage is initiated by DNA glycosylases, which catalyze the excision of oxidized bases, such as 8-hydroxydeoxyguanosine (8-oxodG). Drosophila DNA glycosylases, dOgg1 and RpS3, were ectopically expressed within the mitochondrial matrix in Drosophila S2 cells, causing a severalfold decrease in the levels of 8-oxodG in mitochondrial DNA. Unexpectedly, cells did not show increased resistance to oxidative stress, but instead became more susceptible to treatment with hydrogen peroxide or paraquat. Even in the absence of oxidative challenge, cells expressing RpS3 or dOgg1 in mitochondria exhibited increased apoptosis relative to controls, as determined by flow-cytometric analysis of Annexin V and DNA degradation measured by the Comet assay. Another notable finding was that ectopic expression of either dOgg1 or RpS3 in mitochondria increased cell survival after exposure to the nitric oxide donor SNAP. These results suggest that ectopic expression of one of the constituents of the DNA repair system in mitochondria may cause a perturbation in the base excision repair pathway and lower, rather than enhance, survivability.

Methylation of cytosine to 5-methylcytosine (5mC) is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing. Active demethylation in animals is believed to proceed by DNA glycosylase removal of deaminated or oxidized 5mC. In plants, 5mC is removed from the genome directly by the DEMETER (DME) family of DNA glycosylases. Arabidopsis thaliana DME excises 5mC to activate expression of maternally imprinted genes. Although the related Repressor of Silencing 1 (ROS1) enzyme has been characterized, the molecular basis for 5mC recognition by DME has not been investigated. Here, we present a structure-function analysis of DME and the related DME-like 3 (DML3) glycosylases for 5mC and its oxidized derivatives. Relative to 5mC, DME and DML3 exhibited robust activity toward 5-hydroxymethylcytosine, limited activity for 5-carboxylcytosine, and no activity for 5-formylcytosine. We used homology modeling and mutational analysis of base excision and DNA binding to identify residues important for recognition of 5mC within the context of DNA and inside the enzyme active site. Our results indicate that the 5mC binding pocket is composed of residues from discrete domains and is responsible for discrimination against 5mC derivatives, and suggest that DME, ROS1, and DML3 utilize subtly different mechanisms to probe the DNA duplex for cytosine modifications.

Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

Efficient γ-herpesvirus lytic phase replication requires a virally encoded UNG-type uracil-DNA glycosylase as a structural element of the viral replisome. Uniquely, γ-herpesvirus UNGs carry a seven or eight residue insertion of variable sequence in the otherwise highly conserved minor-groove DNA binding loop. In Epstein-Barr Virus [HHV-4] UNG, this motif forms a disc-shaped loop structure of unclear significance. To ascertain the biological role of the loop insertion, we determined the crystal structure of Kaposi's sarcoma-associated herpesvirus [HHV-8] UNG (kUNG) in its product complex with a uracil-containing dsDNA, as well as two structures of kUNG in its apo state. We find the disc-like conformation is conserved, but only when the kUNG DNA-binding cleft is occupied. Surprisingly, kUNG uses this structure to flip the orphaned partner base of the substrate deoxyuridine out of the DNA duplex while retaining canonical UNG deoxyuridine-flipping and catalysis. The orphan base is stably posed in the DNA major groove which, due to DNA backbone manipulation by kUNG, is more open than in other UNG-dsDNA structures. Mutagenesis suggests a model in which the kUNG loop is pinned outside the DNA-binding cleft until DNA docking promotes rigid structuring of the loop and duplex nucleotide flipping, a novel observation for UNGs.