Isolating Contributions from Intersegmental Transfer to DNA Searching by Alkyladenine DNA Glycosylase

Large genomes pose a challenge to DNA repair pathways because rare sites of damage must be efficiently located from among a vast excess of undamaged sites. Human alkyladenine DNA glycosylase (AAG) employs nonspecific DNA binding interactions and facilitated diffusion to conduct a highly redundant search of adjacent sites. This ensures that every site is searched, but could be a detriment if the protein is trapped in a local segment of DNA. Intersegmental transfer between DNA segments that are transiently in close proximity provides an elegant solution that balances global and local searching processes. It has been difficult to detect intersegmental transfer experimentally; therefore, we developed biochemical assays that allowed us to observe and measure the rates of intersegmental transfer by AAG. AAG has a flexible amino terminus that tunes its affinity for nonspecific DNA, but we find that it is not required for intersegmental transfer. As AAG has only a single DNA binding site, this argues against the bridging model for intersegmental transfer. The rates of intersegmental transfer are strongly dependent on the salt concentration, supporting a jumping mechanism that involves microscopic dissociation and capture by a proximal DNA site. As many DNA-binding proteins have only a single binding site, jumping may be a common mechanism for intersegmental transfer.     

Substrate binding pocket residues of human alkyladenine-DNA glycosylase critical for methylating agent survival

Human alkyladenine-DNA glycosylase (AAG) initiates base excision repair (BER) of alkylated and deaminated bases in DNA. Here, we assessed the mutability of the AAG substrate binding pocket, and the essentiality of individual binding pocket amino acids for survival of methylation damage. We used oligonucleotide-directed mutagenesis to randomize 19 amino acids, 8 of which interact with substrate bases, and created more than 4.5 million variants. We expressed the mutant AAGs in repair-deficient Escherichia coli and selected for protection against the cytotoxicity of either methylmethane sulfonate (MMS) or methyl-lexitropsin (Me-lex), an agent that produces 3-methyladenine as the predominant base lesion. Sequence analysis of 116 methylation-resistant mutants revealed no substitutions for highly conserved Tyr(127)and His(136). In contrast, one mutation, L180F, was greatly enriched in both the MMS- and Me-lex-resistant libraries. Expression of the L180F single mutant conferred 4.4-fold enhanced survival at the high dose of MMS used for selection. The homogeneous L180F mutant enzyme exhibited 2.2-fold reduced excision of 3-methyladenine and 7.3-fold reduced excision of 7-methylguanine from methylated calf thymus DNA. Decreased excision of methylated bases by the mutant glycosylase could promote survival at high MMS concentrations, where the capacity of downstream enzymes to process toxic BER intermediates may be saturated. The mutant also displayed 6.6- and 3.0-fold reduced excision of 1,N(6)-ethenoadenine and hypoxanthine from oligonucleotide substrates, respectively, and a 1.7-fold increase in binding to abasic site-containing DNA. Our work provides in vivo evidence for the substrate binding mechanism deduced from crystal structures, illuminates the function of Leu(180) in wild-type human AAG, and is consistent with a role for balanced expression of BER enzymes in damage survival.     

Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision

The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1.     

Structural basis for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4-ethenocytosine-containing DNA

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.     

The efficiency of hypoxanthine excision by alkyladenine DNA glycosylase is altered by changes in nearest neighbor bases

Alkyladenine DNA glycosylase (AAG) excises a structurally diverse group of damaged purines including hypoxanthine, 1,N(6)-ethenoadenine, 3-methyladenine, and 7-methylguanine from DNA to initiate base excision repair at these sites. Excision occurs in an enzyme.DNA complex in which the damaged base is flipped out of the DNA helix into the enzyme active site. To determine whether local DNA sequence could affect the overall efficiency of excision of hypoxanthine from DNA, single-turnover kinetics of excision, AAG.DNA binding, and melting temperatures were measured for DNA substrates that differed in the base pairs immediately 5' and 3' to hypoxanthine. When Hx was flanked by a 5'G and a 3'C, the efficiency of excision was reduced dramatically in comparison to a duplex containing a 5'T and 3'A. The reduction in excision efficiency was largely due to a decrease in binding affinity of AAG for DNA. The overall effect of GC versus TA nearest neighbors was to magnify the difference in the efficiencies of excision of Hx from pairs with thymine and difluorotoluene from a factor of 5 to a factor of about 100. In general, DNA substrates that were more stable as indicated by higher melting temperatures gave reduced efficiencies of excision of Hx. These results are discussed in terms of a model in which the relative stabilities of base-flipped versus unflipped complexes contribute the overall efficiency of excision and substrate specificity of AAG.     

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB)

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.     

Human alkyladenine DNA glycosylase uses acid-base catalysis for selective excision of damaged purines

Human alkyladenine DNA glycosylase (AAG) protects against alkylative and oxidative DNA damage, flipping damaged nucleotides out of double-stranded DNA and catalyzing the hydrolytic cleavage of the N-glycosidic bond to release the damaged nucleobase. The crystal structure of AAG bound to a DNA substrate reveals features of the active site that could discriminate between oxidatively damaged or alkylated purines and normal purines. A water molecule bound in the active site adjacent to the anomeric carbon of the N-glycosidic bond is suggestive of direct attack by water, with Glu125 acting as a general base. However, biochemical evidence for this proposed mechanism has been lacking. The structure also fails to explain why smaller pyrimidine nucleosides are excluded as substrates from this relatively permissive active site that catalyzes the excision of a structurally diverse group of damaged purine bases. We have used pH dependencies, site-directed mutagenesis, and a variety of substrates to investigate the catalytic mechanism of AAG. Single-turnover excision of hypoxanthine and 1,N(6)-ethenoadenine follows bell-shaped pH-rate profiles, indicating that AAG-catalyzed excision of these neutral lesions requires the action of both a general acid and a general base. In contrast, the pH-rate profile for the reaction of 7-methylguanine, a positively charged substrate, shows only a single ionization corresponding to a general base. These results suggest that AAG activates neutral lesions by protonation of the nucleobase leaving group. Glu125 must be deprotonated in the active form of the enzyme, consistent with a role as a general base that activates and positions a water nucleophile. Acid-base catalysis can account for much of the 10(8)-fold rate enhancement that is achieved by AAG in the excision of hypoxanthine. The prominent role of nucleobase protonation in catalysis by AAG provides a rationale for its specialization toward damaged purines while effectively excluding pyrimidines.     

Defining the functional footprint for recognition and repair of deaminated DNA

Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein–DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.     

Substitution of active site tyrosines with tryptophan alters the free energy for nucleotide flipping by human alkyladenine DNA glycosylase

Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N(6)-ethenoadenine (εA), within 4-fold of that of the wild-type enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40-100-fold decreases in the flipping equilibrium relative to wild-type. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in more stable base pairs.     

Human apurinic/apyrimidinic endonuclease is processive

Apurinic/apyrimidinic endonuclease (AP endo) is believed to play a critical role in repair of oxidative damage of DNA and is proposed to initiate repair of most abasic sites in the base excision repair pathway. AP endo makes a single nick 5' to an abasic site in double-stranded DNA. In this study, we investigated whether AP endo locates an abasic site through a processive or a distributive mechanism. We used a linear multi-abasic site substrate (concatemer), synthesized by ligating together identical 25-nucleotide monomeric units (25-mers). We first determined that the 25-mer monomer from which the concatemers were prepared was nicked by AP endo in a fashion similar to that of the previously published 49-mer substrate with a different sequence. Steady state parameters K(m) and k(cat) and single-turnover parameters for substrate binding were comparable to previously published values. Using the multi-abasic site concatemer, we demonstrated that AP endo was capable of cleaving approximately seven to eight abasic sites, traveling at least 200 nucleotides, before dissociating from its substrate. Thus, AP endo, like uracil DNA glycosylase, behaves in a quasi processive fashion. Processivity could be separated from catalysis, since processivity was maximal at 25 mM NaCl, while the rate of cleavage was maximal at 125 mM salt. In short, nicking activity was maximized close to physiological salt molarities while processivity was midrange at physiological salt concentrations. The latter is likely to be subject to tight regulation by small changes in ionic strength.     

Searching for DNA lesions: structural evidence for lower- and higher-affinity DNA binding conformations of human alkyladenine DNA glycosylase

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.     

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.     

Base-excision repair of oxidative DNA damage by DNA glycosylases

Oxidative damage to DNA caused by free radicals and other oxidants generate base and sugar damage, strand breaks, clustered sites, tandem lesions and DNA-protein cross-links. Oxidative DNA damage is mainly repaired by base-excision repair in living cells with the involvement of DNA glycosylases in the first step and other enzymes in subsequent steps. DNA glycosylases remove modified bases from DNA, generating an apurinic/apyrimidinic (AP) site. Some of these enzymes that remove oxidatively modified DNA bases also possess AP-lyase activity to cleave DNA at AP sites. DNA glycosylases possess varying substrate specificities, and some of them exhibit cross-activity for removal of both pyrimidine- and purine-derived lesions. Most studies on substrate specificities and excision kinetics of DNA glycosylases were performed using oligonucleotides with a single modified base incorporated at a specific position. Other studies used high-molecular weight DNA containing multiple pyrimidine- and purine-derived lesions. In this case, substrate specificities and excision kinetics were found to be different from those observed with oligonucleotides. This paper reviews substrate specificities and excision kinetics of DNA glycosylases for removal of pyrimidine- and purine-derived lesions in high-molecular weight DNA.     

Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells

Using siRNA technology, we down-regulated in human B-lymphoblastoid TK6 cells the two major oxidative DNA glycosylases/AP lyases that repair free radical-induced base damages, hNTH1 and hOGG1. The down-regulation of hOGG1, the DNA glycosylase whose main substrate is the mutagenic but not cytotoxic 8-oxoguanine, resulted in reduced radiation cytotoxicity and decreased double strand break (DSB) formation post-irradiation. This supports the idea that the oxidative DNA glycosylases/AP lyases convert radiation-induced clustered DNA lesions into lethal DSBs and is in agreement with our previous finding that overexpression of hNTH1 and hOGG1 in TK6 cells increased radiation lethality, mutant frequency at the thymidine kinase locus and the enzymatic production of DSBs post-irradiation [N. Yang, H. Galick, S.S. Wallace, Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair (Amst) 3 (2004) 1323-1334]. Interestingly, cells deficient in hNTH1, the DNA glycosylase that repairs a major lethal single free radical damage, thymine glycol, were more radiosensitive but at the same time fewer DSBs were formed post-irradiation. These results indicate that hNTH1 plays two roles in the processing of radiation damages: repair of potentially lethal single lesions and generation of lethal DSBs at clustered damage sites. In contrast, in hydrogen peroxide-treated cells where the majority of free radical DNA damages are single lesions, the base excision repair pathway functioned to protect the cells. Here, overexpression of hNTH1 and hOGG1 resulted in reduced cell killing while suppression of glycosylase expression resulted in elevated cell death.     

Mutational analysis of arginine 276 in the leucine-loop of human uracil-DNA glycosylase

Uracil residues are eliminated from cellular DNA by uracil-DNA glycosylase, which cleaves the N-glycosylic bond between the uracil base and deoxyribose to initiate the uracil-DNA base excision repair pathway. Co-crystal structures of the core catalytic domain of human uracil-DNA glycosylase in complex with uracil-containing DNA suggested that arginine 276 in the highly conserved leucine intercalation loop may be important to enzyme interactions with DNA. To investigate further the role of Arg(276) in enzyme-DNA interactions, PCR-based codon-specific random mutagenesis, and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg(276). All of the R276X mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein in vitro, indicating that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of the R276X preparations was reduced; the least active mutant, R276E, exhibited 0.6% of wildtype activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding studies utilizing a 2-aminopurine deoxypseudouridine DNA substrate showed that all R276X mutants displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276X mutants to single-stranded DNA was much less compromised. Using a concatemeric [(32)P]U.A DNA polynucleotide substrate to assess enzyme processivity, human uracil-DNA glycosylase was shown to use a processive search mechanism to locate successive uracil residues, and Arg(276) mutations did not alter this attribute.     

Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase

Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (k(st)) and spontaneous N-glycosidic bond hydrolysis (k(non)) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (k(st)/k(non) = 10(8)), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.     

Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends

The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.     

Direct repair of 3,N(4)-ethenocytosine by the human ALKBH2 dioxygenase is blocked by the AAG/MPG glycosylase

Exocyclic ethenobases are highly mutagenic DNA lesions strongly implicated in inflammation and vinyl chloride-induced carcinogenesis. While the alkyladenine DNA glycosylase, AAG (or MPG), binds the etheno lesions 1,N⁶-ethenoadenine (?A) and 3,N⁴-ethenocytosine (?C) with high affinity, only ?A can be excised to initiate base excision repair. Here, we discover that the human AlkB homolog 2 (ALKBH2) dioxygenase enzyme catalyzes direct reversal of ?C lesions in both double- and single-stranded DNA with comparable efficiency to canonical ALKBH2 substrates. Notably, we find that in vitro, the non-enzymatic binding of AAG to ?C specifically blocks ALKBH2-catalyzed repair of ?C but not that of methylated ALKBH2 substrates. These results identify human ALKBH2 as a repair enzyme for mutagenic ?C lesions and highlight potential consequences for substrate-binding overlap between the base excision and direct reversal DNA repair pathways.     

Uracil‐DNA glycosylases—Structural and functional perspectives on an essential family of DNA repair enzymes

Uracil‐DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil‐DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.     

Binding of specific DNA base-pair mismatches by N-methylpurine-DNA glycosylase and its implication in initial damage recognition

Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil-DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N(6)-ethenoadenine, varepsilonA) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket.