Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication.

Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox cofactor in biology. Using DNA electrochemistry, we find that binding of the DNA polyanion promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to a ∼500-fold increase in DNA-binding affinity for the oxidized [4Fe4S]3+ cluster versus the reduced [4Fe4S]2+ cluster. This redox switch can be activated from a distance using DNA charge transport (DNA CT) chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated, with possible roles for the redox switch highlighted. A model is described where repair proteins may signal one another using DNA-mediated charge transport as a first step in their search for lesions. The redox switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA polymerase δ, the redox switch provides a means to modulate replication in response to oxidative stress. We thus describe redox signaling interactions of DNA-processing [4Fe4S] enzymes, as well as the most interesting potential players to consider in delineating new DNA-mediated redox signaling networks.

Eukaryotic Base Excision Repair: New Approaches Shine Light on Mechanism.

Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA.

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair.

Structural and biochemical insights into NEIL2's preference for abasic sites.

Cellular DNA is subject to damage from a multitude of sources and repair or bypass of sites of damage utilize an array of context or cell cycle dependent systems. The recognition and removal of oxidatively damaged bases is the task of DNA glycosylases from the base excision repair pathway utilizing two structural families that excise base lesions in a wide range of DNA contexts including duplex, single-stranded and bubble structures arising during transcription. The mammalian NEIL2 glycosylase of the Fpg/Nei family excises lesions from each of these DNA contexts favoring the latter two with a preference for oxidized cytosine products and abasic sites. We have determined the first liganded crystal structure of mammalian NEIL2 in complex with an abasic site analog containing DNA duplex at 2.08 Å resolution. Comparison to the unliganded structure revealed a large interdomain conformational shift upon binding the DNA substrate accompanied by local conformational changes in the C-terminal domain zinc finger and N-terminal domain void-filling loop necessary to position the enzyme on the DNA. The detailed biochemical analysis of NEIL2 with an array of oxidized base lesions indicates a significant preference for its lyase activity likely to be paramount when interpreting the biological consequences of variants.

Theoretical Insights into MutY Glycosylase DNA Repair Mechanism.

Maintaining the integrity of the genome is fundamental to living organisms. To this end, nature developed several mechanisms to find and promptly repair DNA lesions. Among them, base excision repair (BER) enzymes evolved to efficiently carry out this task. Notably, the mechanisms allowing these proteins to search for, detect, and fix DNA damage on a biologically relevant time scale still remain partially unclear. By taking MutY, a BER enzyme implied in the repair of the 8-oxoguanine-adenine mismatches, as a model system, we shed some light on the repair mechanism through a theoretical-computational approach. First, we estimated the effect of the oxidation state of the MutY iron-sulfur cluster on the protein-DNA binding. Then, the redox thermodynamics of both the protein cluster and DNA nucleobases are calculated. Finally, the charge migration kinetics along the double strand bound to the enzyme has been evaluated. The rationalization of our results indicates that the search for DNA lesions is essentially dictated by the redox chemistry of the species involved, i.e., the iron-sulfur redox cofactor and the DNA bound to the enzyme.

Emerging Roles of DNA Glycosylases and the Base Excision Repair Pathway.

The base excision repair (BER) pathway historically has been associated with maintaining genome integrity by eliminating nucleobases with small chemical modifications. In the past several years, however, BER was found to play additional roles in genome maintenance and metabolism, including sequence-specific restriction modification and repair of bulky adducts and interstrand crosslinks. Central to this expanded biological utility are specialized DNA glycosylases - enzymes that selectively excise damaged, modified, or mismatched nucleobases. In this review we discuss the newly identified roles of the BER pathway and examine the structural and mechanistic features of the DNA glycosylases that enable these functions.

A Kinase and a Glycosylase Catabolize Pseudouridine in the Peroxisome to Prevent Toxic Pseudouridine Monophosphate Accumulation.

Pseudouridine (Ψ) is a frequent nucleoside modification that occurs in both noncoding RNAs and mRNAs. In pseudouridine, C5 of uracil is attached to the Rib via an unusual C-glycosidic bond. This RNA modification is introduced on the RNA by site-specific transglycosylation of uridine (U), a process mediated by pseudouridine synthases. RNA is subject to constant turnover, releasing free pseudouridine, but the metabolic fate of pseudouridine in eukaryotes is unclear. Here, we show that in Arabidopsis (Arabidopsis thaliana), pseudouridine is catabolized in the peroxisome by (1) a pseudouridine kinase (PUKI) from the PfkB family that generates 5'-pseudouridine monophosphate (5'-ΨMP) and (2) a ΨMP glycosylase (PUMY) that hydrolyzes ΨMP to uracil and ribose-5-phosphate. Compromising pseudouridine catabolism leads to strong pseudouridine accumulation and increased ΨMP content. ΨMP is toxic, causing delayed germination and growth inhibition, but compromising pseudouridine catabolism does not affect the Ψ/U ratios in RNA. The bipartite peroxisomal PUKI and PUMY are conserved in plants and algae, whereas some fungi and most animals (except mammals) possess a PUMY-PUKI fusion protein, likely in mitochondria. We propose that vacuolar turnover of ribosomal RNA produces most of the pseudouridine pool via 3'-ΨMP, which is imported through the cytosol into the peroxisomes for degradation by PUKI and PUMY, a process involving a toxic 5'-ΨMP intermediate.

Structure of the mammalian adenine DNA glycosylase MUTYH: insights into the base excision repair pathway and cancer.

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9-Rad1-Hus1 complex (9-1-1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9-1-1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function.

Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.

Target search and recognition mechanisms of glycosylase AlkD revealed by scanning FRET-FCS and Markov state models.

DNA glycosylase is responsible for repairing DNA damage to maintain the genome stability and integrity. However, how glycosylase can efficiently and accurately recognize DNA lesions across the enormous DNA genome remains elusive. It has been hypothesized that glycosylase translocates along the DNA by alternating between a fast but low-accuracy diffusion mode and a slow but high-accuracy mode when searching for DNA lesions. However, the slow mode has not been successfully characterized due to the limitation in the spatial and temporal resolutions of current experimental techniques. Using a newly developed scanning fluorescence resonance energy transfer (FRET)-fluorescence correlation spectroscopy (FCS) platform, we were able to observe both slow and fast modes of glycosylase AlkD translocating on double-stranded DNA (dsDNA), reaching the temporal resolution of microsecond and spatial resolution of subnanometer. The underlying molecular mechanism of the slow mode was further elucidated by Markov state model built from extensive all-atom molecular dynamics simulations. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. Furthermore, the essential role of Y27 in AlkD diffusion dynamics was identified both experimentally and computationally. Our results provided mechanistic insights on how conformational dynamics of AlkD-dsDNA complex coordinate different diffusion modes to accomplish the search for DNA lesions with high efficiency and accuracy. We anticipate that the mechanism adopted by AlkD to search for DNA lesions could be a general one utilized by other glycosylases and DNA binding proteins.

The role of cysteines in the structure and function of OGG1.

8-Oxoguanine glycosylase (OGG1) is a base excision repair enzyme responsible for the recognition and removal of 8-oxoguanine, a commonly occurring oxidized DNA modification. OGG1 prevents the accumulation of mutations and regulates the transcription of various oxidative stress-response genes. In addition to targeting DNA, oxidative stress can affect proteins like OGG1 itself, specifically at cysteine residues. Previous work has shown that the function of OGG1 is sensitive to oxidants, with the cysteine residues of OGG1 being the most likely site of oxidation. Due to the integral role of OGG1 in maintaining cellular homeostasis under oxidative stress, it is important to understand the effect of oxidants on OGG1 and the role of cysteines in its structure and function. In this study, we investigate the role of the cysteine residues in the function of OGG1 by mutating and characterizing each cysteine residue. Our results indicate that the cysteines in OGG1 fall into four functional categories: those that are necessary for (1) glycosylase activity (C146 and C255), (2) lyase activity (C140S, C163, C241, and C253), and (3) structural stability (C253) and (4) those with no known function (C28 and C75). These results suggest that under conditions of oxidative stress, cysteine can be targeted for modifications, thus altering the response of OGG1 and affecting its downstream cellular functions.

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity.

DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC.

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

Development of QM/MM (ABEEM polarizable force field) method to simulate the excision reaction mechanism of damaged cytosine.

DNA damages are regarded as having harmful effects on cell. The base excision repair mechanism combats these effects by removing damaged bases. The deglycosylation mechanism of excising damaged bases by DNA glycosylase and the state of the leaving base have been controversial. The enzymatic reaction of DNA glycosylase to remove the damaged bases involves not only the formation and breaking of chemical bonds, but also complex polarization effect and charge transfer, which cannot be accurately simulated by the QM/MM method combined with the fixed charge force field. This work has developed the ABEEM fluctuating polarizable force field combining with the QM method, that is (QM/MM[ABEEM]), to accurately simulate the proton transfer, charge transfer and the charge distribution. The piecewise function is used as the valence-state electronegativity in the QM/MM (ABEEM) to realize the accurate fitting of the charge distribution in reaction. And the charge transfer is accurately simulated by the local charge conservation conditions. Four deglycosylation mechanisms including the monofunctional and difunctional mechanisms of four neutral and protonated cytosine derivatives are explored. It is confirmed that the monofunctional mechanism of Asp-activated nucleophile water is a better deglycosylation mechanism and the base is protonated before the reaction occurs. Protonization of the base reduced the activation energy by 10.00-17.00 kcal/mol. Asp provides the necessary charge for the reaction, and DNA glycosylase preferentially cleaves ɛC. This work provides a theoretical basis for the research of excising damaged bases by DNA glycosylase.

DNA Deformation Exerted by Regulatory DNA-Binding Motifs in Human Alkyladenine DNA Glycosylase Promotes Base Flipping.

Human alkyladenine DNA glycosylase (AAG) is a key enzyme that corrects a broad range of alkylated and deaminated nucleobases to maintain genomic integrity. When encountering the lesions, AAG adopts a base-flipping strategy to extrude the target base from the DNA duplex to its active site, thereby cleaving the glycosidic bond. Despite its functional importance, the detailed mechanism of such base extrusion and how AAG distinguishes the lesions from an excess of normal bases both remain elusive. Here, through the Markov state model constructed on extensive all-atom molecular dynamics simulations, we find that the alkylated nucleobase (N3-methyladenine, 3MeA) everts through the DNA major groove. Two key AAG motifs, the intercalation and E131-N146 motifs, play active roles in bending/pressing the DNA backbone and widening the DNA minor groove during 3MeA eversion. In particular, the intercalated residue Y162 is involved in buckling the target site at the early stage of 3MeA eversion. Our traveling-salesman based automated path searching algorithm further revealed that a non-target normal adenine tends to be trapped in an exo site near the active site, which however barely exists for a target base 3MeA. Collectively, these results suggest that the Markov state model combined with traveling-salesman based automated path searching acts as a promising approach for studying complex conformational changes of biomolecules and dissecting the elaborate mechanism of target recognition by this unique enzyme.

Distortion of double-stranded DNA structure by the binding of the restriction DNA glycosylase R.PabI.

R.PabI is a restriction DNA glycosylase that recognizes the sequence 5'-GTAC-3' and hydrolyses the N-glycosidic bond of adenine in the recognition sequence. R.PabI drastically bends and unwinds the recognition sequence of double-stranded DNA (dsDNA) and flips the adenine and guanine bases in the recognition sequence into the catalytic and recognition sites on the protein surface. In this study, we determined the crystal structure of the R.PabI-dsDNA complex in which the dsDNA is drastically bent by the binding of R.PabI but the base pairs are not unwound. This structure is predicted to be important for the indirect readout of the recognition sequence by R.PabI. In the complex structure, wedge loops of the R.PabI dimer are inserted into the minor groove of dsDNA to stabilize the deformed dsDNA structure. A base stacking is distorted between the two wedge-inserted regions. R.PabI is predicted to utilize the distorted base stacking for the detection of the recognition sequence.

Nucleosome Core Particles Lacking H2B or H3 Tails Are Altered Structurally and Have Differential Base Excision Repair Fingerprints.

A recently discovered post-translational modification of histone proteins is the irreversible proteolytic clipping of the histone N-terminal tail domains. This modification is involved in the regulation of various biological processes, including the DNA damage response. In this work, we used chemical footprinting to characterize the structural alterations to nucleosome core particles (NCPs) that result from a lack of a histone H2B or H3 tail. We also examine the influence of these histone tails on excision of the mutagenic lesion 1,N6-ethenoadenine (εA) by the repair enzyme alkyladenine DNA glycosylase. We found that the absence of the H2B or H3 tail results in altered DNA periodicity relative to that of native NCPs. We correlated these structural alterations to εA excision by utilizing a global analysis of 21 εA sites in NCPs and unincorporated duplex DNA. In comparison to native NCPs, there is enhanced excision of εA in tailless H2B NCPs in regions that undergo DNA unwrapping. This enhanced excision is not observed for tailless H3 NCPs; rather, excision is inhibited in more static areas of the NCP not prone to unwrapping. Our results support in vivo observations of alkylation damage profiles and the potential role of tail clipping as a mechanism for overcoming physical obstructions caused by packaging in NCPs but also reveal the potential inhibition of repair by tail clipping in some locations. Taken together, these results further our understanding of how base excision repair can be facilitated or diminished by histone tail removal and contribute to our understanding of the underlying mechanism that leads to mutational hot spots.

Recognition of 1,N2-ethenoguanine by alkyladenine DNA glycosylase is restricted by a conserved active-site residue.

The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1,N6-ethenoadenine (ϵA) and 1,N2-ethenoguanine (ϵG). However, it is not clear how AAG might accommodate ϵG in an active site that is complementary to ϵA. This prompted a thorough investigation of AAG-catalyzed excision of ϵG from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ϵG in its natural ϵG·C context is very poorly recognized relative to ϵA·T. Bulged and mispaired ϵG contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ϵG site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ϵG. Consistent with this model, the N169S variant of AAG was 7-fold more active for excision of ϵG compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ϵG is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ϵG lesions.

DNA Sequence Modulates the Efficiency of NEIL1-Catalyzed Excision of the Aflatoxin B1-Induced Formamidopyrimidine Guanine Adduct.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B1 (AFB1) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB1-FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB1-FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB1-FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB1-FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB1-FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB1-FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions.

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge-dipole and CH-π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.