Step-by-step mechanism of DNA damage recognition by human 8-oxoguanine DNA glycosylase

Background

Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids.

Methods

Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA–substrates was examined.

Results

The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated.

Conclusions

The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently.

General significance

The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1.     

The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity.

The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine). Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3. Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5' apurinic/apyrimidinic (AP) sites via a beta-elimination reaction. Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3' incised AP site by a Mg2+-dependent hydrolytic mechanism. In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities. A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5' AP sites. Activity was also detected for the Mg2+-dependent removal of trans -4-hydroxy-2-pentenal-5-phosphate at 3' incised AP sites and from intact AP sites. Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair. However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans -4-hydroxy-2-pentenal-5-phosphate left at a 3' terminus created by a beta-elimination catalyst.     

Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5-methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8-oxoguanine (oxoGua), which is repaired by 8-oxoguanine-DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite-base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.     

Distinct repair activities of human 7,8-dihydro-8-oxoguanine DNA glycosylase and formamidopyrimidine DNA glycosylase for formamidopyrimidine and 7,8-dihydro-8-oxoguanine

7,8-dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and are involved in mutagenic and/or lethal events in cells. Both lesions are repaired by human 7, 8-dihydro-8-oxoguanine DNA glycosylase (hOGG1) and formamidopyrimidine DNA glycosylase (Fpg) in human and Escherichia coli cells, respectively. In the present study, the repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The k(cat)/K(m) values of hOGG1 for 8-oxoG and me-Fapy were comparable, and this was also the case for Fpg. However, the k(cat)/K(m) values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. Analysis of the Schiff base intermediate by NaBH(4) trapping implied that lower substrate affinity and slower hydrolysis of the intermediate for hOGG1 than Fpg accounted for the difference. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8- (hOGG1) and 12-fold (Fpg) between the most and least preferred bases. Surprisingly, such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair. The repair efficiency of me-Fapy randomly incorporated in M13 DNA varied at the sequence level, but orders of preferred and unpreferred repair sites were quite different for hOGG1 and Fpg. The distinctive activities of hOGG1 and Fpg including enzymatic parameters (k(cat)/K(m)), paired base, and sequence context effects may originate from the differences in the inherent architecture of the DNA binding domain and catalytic mechanism of the enzymes.     

Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.     

Separation-of-function mutants unravel the dual-reaction mode of human 8-oxoguanine DNA glycosylase

7,8-Dihydro-8-oxoguanine (8oxoG) is a major mutagenic base lesion formed when reactive oxygen species react with guanine in DNA. The human 8oxoG DNA glycosylase (hOgg1) recognizes and initiates repair of 8oxoG. hOgg1 is acknowledged as a bifunctional DNA glycosylase catalyzing removal of the damaged base followed by cleavage of the backbone of the intermediate abasic DNA (AP lyase/β-elimination). When acting on 8oxoG-containing DNA, these two steps in the hOgg1 catalysis are considered coupled, with Lys249 implicated as a key residue. However, several lines of evidence point to a concurrent and independent monofunctional hydrolysis of the N-glycosylic bond being the in?vivo relevant reaction mode of hOgg1. Here, we present biochemical and structural evidence for the monofunctional mode of hOgg1 by design of separation-of-function mutants. Asp268 is identified as the catalytic residue, while Lys249 appears critical for the specific recognition and final alignment of 8oxoG during the hydrolysis reaction.     

Characterization of an 8-oxoguanine DNA glycosylase from Methanococcus jannaschii.

A thermostable 8-oxoguanine (oxoG) DNA glycosylase from Methanococcus jannaschii has been expressed in Escherichia coli, purified, and characterized. The enzyme, which has been named mjOgg, belongs to the same diverse DNA glycosylase superfamily as the 8-oxoguanine DNA glycosylases from yeast (yOgg1) and human (hOgg1) but is substantially different in sequence. In addition, unlike its eukaryotic counterparts, which have a strong preference for oxoG.C base pairs, mjOgg has little specificity for the base opposite oxoG. mjOgg has both DNA glycosylase and DNA lyase (beta-elimination) activity, and the combined glycosylase/lyase activity occurs at a rate comparable with the glycosylase activity alone. Mutation of Lys-129, analogous to Lys-241 of yOgg1, abolishes glycosylase activity.     

Interactions of human 8-oxoguanine DNA glycosylase with single-and double-stranded DNAs

The interaction of human 8-oxoguanine (8-oxoG) DNA glycosylase (hOGG1) with single-and double-stranded oligodeoxyribonucleotides (ODNs) was studied by a method of stepwise increase in ligand complexity. ODNs were shown to act as competitive inhibitors with respect to the substrate of the reaction catalyzed by hOGG1. K _I was estimated for various homo-and hetero-ODNs. All nucleotides covered by the enzyme globule proved to additively interact with hOGG1. An increase in the ODN size n by one nucleotide or base pair in d(pN)_n and their duplexes monotonically increased their affinity for hOGG1 by a factor of 1.4–1.5 until n = 10, mostly due to weak nonspecific additive contacts between hOGG1 and the sugar-phosphate backbone. Weak nonspecific additive interactions contributed about five orders of magnitude to the total affinity of hOGG1 for specific DNA (K _d ～ 10^?5 M). Specific 8-oxoG increased the affinity of DNA for the enzyme by three orders of magnitude (K _d ～ 10^?8 M). The main features of the recognition of specific DNA by hOGG1 were analyzed.     

Mechanism of recognition and repair of damaged DNA by human 8-oxoguanine DNA glycosylase hOGG1

Recent data on structural and biochemical features of human 8-oxoguanine DNA glycosylase (hOGG1) has enabled detailed evaluation of the mechanism by which the damaged DNA bases are recognized and eliminated from the chain. Pre-steady-state kinetic studies with recording of conformational transitions of the enzyme and DNA substrate significantly contribute to understanding of this mechanism. In this review we particularly focus on the interrelationship between the conformational changes of interacting molecules and kinetics of their interaction and on the nature of each elementary step during the enzymatic process. Exhaustive analysis of these data and detailed mechanism of hOGG1-catalyzed reaction are proposed.     

Nickel(II)-catalysed oxidative guanine and DNA damage beyond 8-oxoguanine

Oxidative DNA damage is one of the most important and most studied mechanisms of disease. It has been associated with a range of terminal diseases such as cancer, heart disease, hepatitis, and HIV, as well as with a variety of everyday ailments. There are various mechanisms by which this type of DNA damage can be initiated, through radiation and chemical oxidation, among others; however, these mechanisms have yet to be fully elucidated. A HPLC-UV-EC study of the oxidation of DNA mediated by nickel(II) obtained results that show an erratic, almost oscillatory formation of 8-oxoguanine (8-oxoG) from free guanine and from guanine in DNA. Sporadic 8-oxoG concentrations were also observed when 8-oxoG alone was subjected to these conditions. A HPLC-MS/MS study showed the formation of oxidised-guanidinohydantoin (oxGH) from free guanine at pH 11, and the formation of guanidinohydantoin (GH) from DNA at pH 5.5.     

Clostridium acetobutylicum 8-oxoguanine DNA glycosylase (Ogg) differs from eukaryotic Oggs with respect to opposite base discrimination

During repair of damaged DNA, the oxidized base 8-oxoguanine (8-oxoG) is removed by 8-oxoguanine-DNA glycosylase (Ogg) in eukaryotes and most archaea, whereas in most bacteria it is removed by formamidopyrimidine-DNA glycosylase (Fpg). We report the first characterization of a bacterial Ogg, Clostridium acetobutylicum Ogg (CacOgg). Like human OGG1 and Escherichia coli Fpg (EcoFpg), CacOgg excised 8-oxoguanine. However, unlike hOGG1 and EcoFpg, CacOgg showed little preference for the base opposite the damage during base excision and removed 8-oxoguanine from single-stranded DNA. Thus, our results showed unambiguous qualitative functional differences in vitro between CacOgg and both hOGG1 and EcoFpg. CacOgg differs in sequence from the eukaryotic enzymes at two sequence positions, M132 and F179, which align with amino acids (R154 and Y203) in human OGG1 (hOGG1) found to be involved in opposite base interaction. To address the sequence basis for functional differences with respect to opposite base interactions, we prepared three CacOgg variants, M132R, F179Y, and M132R/F179Y. All three variants showed a substantial increase in specificity for 8-oxoG.C relative to 8-oxoG.A. While we were unable to definitively associate these qualitative functional differences with differences in selective pressure between eukaryotes, Clostridia, and other bacteria, our results are consistent with the idea that evolution of Ogg function is based on kinetic control of repair.     

Down-regulation of 8-oxoguanine DNA glycosylase 1 expression in the airway epithelium ameliorates allergic lung inflammation

Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits.     

Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis

8-Oxoguanine DNA glycosylase (OGG1) is a major DNA repair enzyme in mammalian cells. OGG1 participates in the repair of 8-oxoG, the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. In this study, antibodies directed against purified recombinant human OGG1 (hOGG1) or murine (mOGG1) protein were chemically conjugated to either the photosensitizer Rose Bengal or the fluorescent dye Texas red. These dye-protein conjugates, in combination with binding assays, were used to identify associations between mOGG1 and the cytoskeleton of NIH3T3 fibroblasts. Results from these binding studies showed that mOGG1 associates with the cytoskeleton by specifically binding to the centriole and microtubules radiating from the centrosome at interphase and the spindle assembly at mitosis. Similar results were obtained with hOGG1. Together results reported in this study suggest that OGG1 is a microtubule-associated protein itself or that OGG1 utilizes yet to be identified motor proteins to ride on microtubules as tracks facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage.     

The kinetic analysis of recognition of the damaged nucleotides by mutant forms of the 8-oxoguanine DNA glycosylase hOGG1

We have investigated the role of Tyr-203, His-270, and Lys-249 amino acid residues from the 8-oxoguanine glycosylase (hOGG1) active site in the process of recognition of 7,8-dihydro-8-oxoguanine (oxoG) damaged nucleotide and in the catalytic stages of enzymatic reaction. The pre-steady state kinetic analysis of conformational transitions of mutant forms of the enzyme and model DNA substrates during the enzymatic process revealed that the studied amino acid residues are involved in the specific binding of DNA substrates. The Tyr-203 is responsible for recognition of the damaged nucleotide; interaction between His-270 and DNA is necessary for the formation of the catalytically active complex with the oxoG-containing DNA. The Lys-249 acts not only as one of the catalytically important amino acids of the active site of the enzyme, but also plays a significant role in the formation of specific enzyme–substrate complex. The present study significantly complements the molecular-kinetic model of the enzymatic reaction and helps to clarify the origin of the high specificity of hOGG1 to oxidized bases in DNA.     

Cardiac overexpression of 8-oxoguanine DNA glycosylase 1 protects mitochondrial DNA and reduces cardiac fibrosis following transaortic constriction

Cardiac failure is associated with increased levels of oxidized DNA, especially mitochondrial (mtDNA). It is not known if oxidized mtDNA contributes to cardiac dysfunction. To test if protection of mtDNA can reduce cardiac injury, we produced transgenic mice with cardiomyocyte-specific overexpression of the DNA repair enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) isoform 2a. In one line of mice, the transgene increased OGG1 activity by 115% in mitochondria and by 28% in nuclei. OGG1 transgenic mice demonstrated significantly lower cardiac mitochondrial levels of the DNA guanine oxidation product 7,8-dihydro-8-oxoguanine (8-oxo-dG) under basal conditions, after doxorubicin administration, or after transaortic constriction (TAC), but the transgene produced no detectable reduction in nuclear 8-oxo-dG content. OGG1 mice were tested for protection from the cardiac effects of TAC 13 wk after surgery. Compared with FVB-TAC mice, hearts from OGG1-TAC mice had lower levels of β-myosin heavy chain mRNA but they did not display significant differences in the ratio of heart weight to tibia length or protection of cardiac function measured by echocardiography. The principle benefit of OGG1 overexpression was a significant decrease in TAC-induced cardiac fibrosis. This protection was indicated by reduced Sirius red staining on OGG1 cardiac sections and by significantly decreased induction of collagen 1 and 3 mRNA expression in OGG1 hearts after TAC surgery. These results provide a new model to assess the damaging cardiac effects of 8-oxo-dG formation and suggest that increased repair of 8-oxo-dG in mtDNA decreases cardiac pathology.     

Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.     

An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana

Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein. The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants. We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsisthaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins. Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate characteristic of all bifunctional DNA glycosylases/lyases. Consistent with its in vitro activity, expression of AtOGG1 suppresses the mutator phenotype of an E. coli strain deficient in 8-oxoG repair. Our results suggest that AtOgg1 is an structural and functional homologue of Ogg1 and establish the presence of two distinct 8-oxoG repair enzymes in Arabidopsis.     

Mammalian 8-oxoguanine DNA glycosylase 1 incises 8-oxoadenine opposite cytosine in nuclei and mitochondria,while a different glycosylase incises 8-oxoadenine opposite guanine in nuclei

Cells are continuously exposed to oxidative species, which cause several types of oxidative DNA lesions. Repair of some of these lesions has been well characterized but little is known about the repair of many DNA lesions. The oxidized adenine base, 7,8-dihydro-8-oxoadenine (8-oxoA), is a relatively common DNA lesion, which is believed to be mutagenic in mammalian cells. This study investigates repair of 8-oxoA in nuclear and mitochondrial mammalian extracts. In nuclei, 8-oxoA:C and 8-oxoA:G base pairs are recognized and cleaved; in contrast, only 8-oxoA:C base pairs are cleaved in mitochondria. High stability of the DNA helix increased the efficiency of incision of 8-oxoA, and the efficiency decreased at DNA bends and condensed regions of the helix. Using liver extracts from mice knocked out for 8-oxoguanine DNA glycosylase 1 (OGG1), we demonstrated that OGG1 is the only glycosylase that incises 8-oxoA, when base-paired with cytosine in mitochondria and nuclei, but a different enzyme incises 8-oxoA when base-paired with guanine in the nucleus. Consistent with this result, a covalent DNA-protein complex was trapped using purified human OGG1 or human nuclear or mitochondrial extracts with a DNA substrate containing an 8-oxoA:C base pair.     

Computational analysis of the mode of binding of 8-oxoguanine to formamidopyrimidine-DNA glycosylase

8-Oxoguanine (8OG) is the most prevalent form of oxidative DNA damage. In bacteria, 8OG is excised by formamidopyrimidine glycosylase (Fpg) as the initial step in base excision repair. To efficiently excise this lesion, Fpg must discriminate between 8OG and an excess of guanine in duplex DNA. In this study, we explore the structural basis underlying this high degree of selectivity. Two structures have been reported in which Fpg is bound to DNA, differing with respect to the position of the lesion in the active site, one structure showing 8OG bound in the syn conformation and the other in the anti conformation. Remarkably, the results of our all-atom simulations are consistent with both structures. The syn conformation observed in the crystallographic structure of Fpg obtained from Bacillus stearothermophilus is stabilized through interaction with E77, a nonconserved residue. Replacement of E77 with Ser, creating the Fpg sequence found in Escherichia coli and other bacteria, results in preferred binding of 8OG in the anti conformation. Our calculations provide novel insights into the roles of active site residues in binding and recognition of 8OG by Fpg.