Kinetic conformational analysis of human 8-oxoguanine-DNA glycosylase

7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base followed by a much slower beta-elimination reaction at the 3'-side of the resulting abasic site. Many features of OGG1 mechanism, including its low beta-elimination activity and high specificity for a cytosine base opposite the lesion, remain poorly explained despite the availability of structural information. In this study, we analyzed the substrate specificity and the catalytic mechanism of OGG1 acting on various DNA substrates using stopped-flow kinetics with fluorescence detection. Combining data on intrinsic tryptophan fluorescence to detect conformational transitions in the enzyme molecule and 2-aminopurine reporter fluorescence to follow DNA dynamics, we defined three pre-excision steps and assigned them to the processes of (i) initial encounter with eversion of the damaged base, (ii) insertion of several enzyme residues into DNA, and (iii) enzyme isomerization to the catalytically competent form. The individual rate constants were derived for all reaction stages. Of all conformational changes, we identified the insertion step as mostly responsible for the opposite base specificity of OGG1 toward 8-oxoG:C as compared with 8-oxoG:T, 8-oxoG:G, and 8-oxoG:A. We also investigated the kinetic mechanism of OGG1 stimulation by 8-bromoguanine and showed that this compound affects the rate of beta-elimination rather than pre-excision dynamics of DNA and the enzyme.     

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

Interactions of human 8-oxoguanine-DNA glycosylase (hOGG1) with single- and double-stranded oligodeoxyribonucleotides (ODN) have been studied by the method of stepwise increase in ligand complexity. The ODNs have been found to inhibit the glycosylase-catalyzed reaction competitively. The K1 values have been determined for a set of ODNs. All units of non-specific DNA within the enzyme footprint have been shown to interact with the protein globule in an additive manner. An increase in the d(pN)n length (n) by one unit caused a monotonous 1.4-1.5-fold increase in their affinity for the glycosylase ODN until n = 10, mostly due to weak nonspecific contacts of the enzyme and the sugar-phosphate backbone. The weak nonspecific additive interactions contributed about five orders of magnitude in the affinity of hOGG1 for specific DNA (Kd approximately 10(-5) M), whereas introduction of a 8-oxoguanine residue added about three orders of magnitude to this affinity (Kd approximately 10(-8) M). Quantitative features of recognition of specific DNA by the enzyme are analyzed.     

Specificity of stimulation of human 8-oxoguanine-DNA glycosylase by AP endonuclease

Human 8-oxoguanine-DNA glycosylase OGG1 is an enzyme that removes abundant oxidative lesion 8-oxoguanine (8-oxoG) from DNA. Excision of 8-oxoG by OGG1 is inhibited by the abasic DNA reaction product and is stimulated by AP endonuclease APEX1. Besides 8-oxoG, OGG1 shows activity towards several other base lesions. Here we report that APEX1 efficiently stimulates OGG1 on good substrates (8-oxoadenine, 8-oxoinosine, or 6-methoxy-8-oxoguanine opposite to cytosine) but the stimulation is low or absent with poor OGG1 substrates (8-oxoadenine or 8-oxoinosine opposite to thymine; 8-oxoG or 8-aminoguanine opposite to adenine; 8-oxonebularine, 8-metoxyguanine, inosine or guanine opposite to cytosine). APEX1 significantly improves the ability of OGG1 to excise 8-aminoguanine from its naturally occurring pair with cytosine, making it possible that OGG1 repairs this lesion. Overall, APEX1 serves to improve specificity of OGG1 for its biologically relevant substrates.     

Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease

Human 8-oxoguanine-DNA glycosylase (OGG1) is the main human base excision protein that removes a mutagenic lesion 8-oxoguanine (8-oxoG) from DNA. Since OGG1 has DNA glycosylase and weak abasic site (AP) lyase activities and is characterized by slow product release, turnover of the enzyme acting alone is low. Recently it was shown that human AP endonuclease (APE1) enhances the activity of OGG1. This enhancement was proposed to be passive, resulting from APE1 binding to or cleavage of AP sites after OGG1 dissociation. Here we present evidence that APE1 could actively displace OGG1 from its product, directly increasing the turnover of OGG1. We have observed that APE1 forms an electrophoretically detectable complex with OGG1 cross-linked to DNA by sodium borohydride. Using oligonucleotide substrates with a single 8-oxoG residue located in their 5'-terminal, central or 3'-terminal part, we have demonstrated that OGG1 activity does not increase only for the first of these three substrates, indicating that APE1 interacts with the DNA stretch 5' to the bound OGG1 molecule. In kinetic experiments, APE1 enhanced the product release constant but not the rate constant of base excision by OGG1. Moreover, OGG1 bound to a tetrahydrofuran analog of an abasic site stimulated the activity of APE1 on this substrate. Using a concatemeric DNA substrate, we have shown that APE1 likely displaces OGG1 in a processive mode, with OGG1 remaining on DNA but sliding away in search for a new lesion. Altogether, our data support a model in which APE1 specifically recognizes an OGG1/DNA complex, distorts a stretch of DNA 5' to the OGG1 molecule, and actively displaces the glycosylase from the lesion.     

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG.     

A liquid chromatography-mass spectrometric assay for measuring activity of human 8-oxoguanine-DNA glycosylase

A new assay for measuring glycosylase activity of human 8-oxoguanine-DNA glycosylase is described. The assay measures the amount of released 8-oxoguanine from synthetic oligonucleotides containing modified base in the middle of the sequence. After enzymatic release, the amount of base is quantified by liquid chromatography-mass spectrometry. Chromatographic separation is carried out on a reversed-phase C₁₈ column using 10% methanol/water. Quantitation of 8-oxoguanine is carried out by negative-ion electrospray on a single quadrupole mass spectrometer operated in selected-ion monitoring mode. The limit of quantitation was 6 nM and the assay was linear from 6 to 1000 nM. The method was evaluated by monitoring the kinetics of base excision of several substrates as well as by measuring stimulation of activity in the presence of APE1 endonuclease. The new assay provides much higher throughput compared to traditional gel-based assays, which is particularly important when large number of samples need to analyzed.     

Substrate specificity and reaction mechanism of murine 8-oxoguanine-DNA glycosylase

Genomic DNA is prone to oxidation by reactive oxygen species. A major product of DNA oxidation is the miscoding base 8-oxoguanine (8-oxoG). The mutagenic effects of 8-oxoG in mammalian cells are prevented by a DNA repair system consisting of 8-oxoguanine-DNA glycosylase (Ogg1), adenine-DNA glycosylase, and 8-oxo-dGTPase. We have cloned, overexpressed, and characterized mOgg1, the product of the murine ogg1 gene. mOgg1 is a DNA glycosylase/AP lyase belonging to the endonuclease III family of DNA repair enzymes. The AP lyase activity of mOgg1 is significantly lower than its glycosylase activity. mOgg1 releases 8-oxoG from DNA when paired with C, T, or G, but efficient DNA strand nicking is observed only with 8-oxoG:C. Binding of mOgg1 to oligonucleotides containing 8-oxoG:C is strong (K(D) = 51.5 nm), unlike other mispairs. The average residence time for mOgg1 bound to substrate containing 8-oxoG:C is 18.3 min; the time course for accumulation of the NaBH(4)-sensitive intermediate suggests a two-step reaction mechanism. Various analogs of 8-oxoG were tested as substrates for mOgg1. An electron-withdrawing or hydrogen bond acceptor moiety at C8 is required for efficient binding of mOgg1. A substituent at C6 and a keto group at C8 are required for cleavage. The proposed mechanism of 8-oxoG excision involves protonation of O(8) or the deoxyribose oxygen moiety.     

Acetylation of human 8-oxoguanine-DNA glycosylase by p300 and its role in 8-oxoguanine repair in vivo

The human 8-oxoguanine-DNA glycosylase 1 (OGG1) is the major DNA glycosylase responsible for repair of 7,8-dihydro-8-oxoguanine (8-oxoG) and ring-opened fapyguanine, critical mutagenic DNA lesions that are induced by reactive oxygen species. Here we show that OGG1 is acetylated by p300 in vivo predominantly at Lys338/Lys341. About 20% of OGG1 is present in acetylated form in HeLa cells. Acetylation significantly increases OGG1's activity in vitro in the presence of AP-endonuclease by reducing its affinity for the abasic (AP) site product. The enhanced rate of repair of 8-oxoG in the genome by wild-type OGG1 but not the K338R/K341R mutant, ectopically expressed in oxidatively stressed OGG1-null mouse embryonic fibroblasts, suggests that acetylation increases OGG1 activity in vivo. At the same time, acetylation of OGG1 was increased by about 2.5-fold after oxidative stress with no change at the polypeptide level. OGG1 interacts with class I histone deacetylases, which may be responsible for its deacetylation. Based on these results, we propose a novel regulatory function of OGG1 acetylation in repair of its substrates in oxidatively stressed cells.     

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the k_cat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The k_cat values for substrates of different sequences correlate with the DNA twist, while the K_M values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the K_M and k_cat values for different sequences containing oxoG were found.     

Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent k_cat of 0.1 min^–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ~5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (K_d ~ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (K_d ~ 23.4 nM) and the affinity for its final β-elimination product was much lower (K_d ~ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.     

Age-dependent changes in 8-oxoguanine-DNA glycosylase activity are modulated by adaptive responses to physical exercise in human skeletal muscle

8-Oxo-7,8-dihydroguanine (8-oxoG) accumulates in the genome over time and is believed to contribute to the development of aging characteristics of skeletal muscle and various aging-related diseases. Here, we show a significantly increased level of intrahelical 8-oxoG and 8-oxoguanine-DNA glycosylase (OGG1) expression in aged human skeletal muscle compared to that of young individuals. In response to exercise, the 8-oxoG level was lastingly elevated in sedentary young and old subjects, but returned rapidly to preexercise levels in the DNA of physically active individuals independent of age. 8-OxoG levels in DNA were inversely correlated with the abundance of acetylated OGG1 (Ac-OGG1), but not with total OGG1, apurinic/apyrimidinic endonuclease 1 (APE1), or Ac-APE1. The actual Ac-OGG1 level was linked to exercise-induced oxidative stress, as shown by changes in lipid peroxide levels and expression of Cu,Zn-SOD, Mn-SOD, and SIRT3, as well as the balance between acetyltransferase p300/CBP and deacetylase SIRT1, but not SIRT6 expression. Together these data suggest that that acetylated form of OGG1, and not OGG1 itself, correlates inversely with the 8-oxoG level in the DNA of human skeletal muscle, and the Ac-OGG1 level is dependent on adaptive cellular responses to physical activity, but is age independent.     

Human 8-oxoguanine-DNA glycosylase 1 protein and gene are expressed more abundantly in the superficial than basal layer of human epidermis

Human 8-oxoguanine-DNA glycosylase 1 (hOGG1) repairs 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) which results from oxidation of guanine. Reactive oxygen species (ROS) formed in response to ultraviolet (UV) radiation cause this DNA damage, which is involved in pathological processes such as carcinogenesis and aging. The initiation of skin tumors probably requires penetration of UV to the actively dividing basal layer of the epidermis in order for acute damage to become fixed as mutations. Previously, the majority of UVB fingerprint mutations have been found in the upper layers of human skin tumors, while UVA mutations have been found mostly in the lower layer. Our aim was to determine whether this localization of UVA-induced DNA damage is related to stratification of the repair-enzyme hOGG1. Anti-hOGG1 immunohistochemical staining of frozen sections of human foreskin, adult buttock skin, and reconstructed human skin samples showed the highest expression of hOGG1 in the superficial epidermal layer (stratum granulosum). Study of the hOGG1 mRNA expression again showed the highest level in the upper region of the epidermis. This was not regulated by UV irradiation but by the differentiation state of keratinocytes as calcium-induced differentiation increased hOGG1 gene expression. UVA-induced 8-oxo-dG was repaired more rapidly in the upper layer of human skin compared to the lower layers. Our results indicate that weaker expression of the nuclear form of hOGG1 enzyme in the basal cells of the epidermis may lead to a lack of DNA repair in these cells and therefore accumulation of UVA-induced oxidative DNA mutations.     

Inactivation of mammalian 8-oxoguanine-DNA glycosylase by cadmium(II): implications for cadmium genotoxicity

Cadmium(II) is a toxic, mutagenic and carcinogenic metal (IARC Class 1 human carcinogen). It causes damage to eukaryotic cells both in acute and chronic modes of exposure via multiple biochemical mechanisms. In particular, Cd diminishes the capacity of cells to repair oxidative DNA damage. Oxidative DNA lesions are important precursors to mutations and ultimately may lead to neoplastic transformation of human cells. We investigated interactions of Cd with murine Ogg1 (mOgg1), an enzyme that removes 8-oxoguanine (8-oxoG), an abundant oxidative lesion, from DNA. Cd(2+) and Zn(2+), but not other divalent cations tested, suppressed mOgg1-catalyzed reactions. The apparent inhibition by Cd consisted of at least two independent processes: irreversible, DNA-independent first-order inactivation of mOgg1 and DNA-dependent inhibition. Irreversibly inactivated mOgg1 has nearly normal affinity for damaged DNA and a normal catalytic rate constant but is defective in formation of the covalent reaction intermediate. When both modes of inhibition are in effect, the catalytic rate constant is dramatically lowered, while affinity to damaged DNA is decreased moderately. Potential sites for Cd binding in mOgg1 and mOgg1-DNA complex are identified. Inactivation of Ogg1 may play a role in the mutagenic and carcinogenic action of Cd.     

Structural and functional determinants of the archaeal 8-oxoguanine-DNA glycosylase AGOG for DNA damage recognition and processing

8-Oxoguanine (GO) is a major purine oxidation product in DNA. Because of its highly mutagenic properties, GO absolutely must be eliminated from DNA. To do this, aerobic and anaerobic organisms from the three kingdoms of life have evolved repair mechanisms to prevent its deleterious effect on genetic integrity. The major way to remove GO is the base excision repair pathway, usually initiated by a GO-DNA glycosylase. First identified in bacteria (Fpg) and eukaryotes (OGG1), GO-DNA glycosylases were more recently identified in archaea (OGG2 and AGOG). AGOG is the less documented enzyme and its mode of damage recognition and removing remains to be clarified at the molecular and atomic levels. This study presents a complete structural characterisation of apo AGOGs from Pyrococcus abyssi (Pab) and Thermococcus gammatolerans (Tga) and the first structure of Pab-AGOG bound to lesion-containing single- or double-stranded DNA. By combining X-ray structure analysis, site directed mutagenesis and biochemistry experiments, we identified key amino acid residues of AGOGs responsible for the specific recognition of the lesion and the base opposite the lesion and for catalysis. Moreover, a unique binding mode of GO, involving double base flipping, never observed for any other DNA glycosylases, is revealed. In addition to unravelling the properties of AGOGs, our study, through comparative biochemical and structural analysis, offers new insights into the evolutionary plasticity of DNA glycosylases across all three kingdoms of life.     

Two sequential phosphates 3' adjacent to the 8-oxoguanosine are crucial for lesion excision by E. coli Fpg protein and human 8-oxoguanine-DNA glycosylase

Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are base excision repair enzymes involved in the 8-oxoguanine (oxoG) repair pathway. Specific contacts between these enzymes and DNA phosphate groups play a significant role in DNA-protein interactions. To reveal the phosphates crucial for lesion excision by Fpg and hOGG1, modified DNA duplexes containing pyrophosphate and OEt-substituted pyrophosphate internucleotide (SPI) groups near the oxoG were tested as substrate analogues for both proteins. We have shown that Fpg and hOGG1 recognize and specifically bind the DNA duplexes tested. We have found that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the 8-oxoguanosine (oxodG) and one nucleotide 3' away from it. In contrast, they efficiently incised DNA duplexes bearing the same phosphate modifications 5' to the oxodG and two nucleotides 3' away from the lesion. The effect of these phosphate modifications on the substrate properties of oxoG-containing DNA duplexes is discussed. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or SPI groups immediately 3' to the oxodG or one nucleotide 3' away from it are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a wild-type enzymes bound to oxoG-containing DNA.     

Correlated cleavage of damaged DNA by bacterial and human 8-oxoguanine-DNA glycosylases

Many enzymes acting on specific rare lesions in DNA are suggested to search for their targets by facilitated one-dimensional diffusion. We have used a recently developed correlated cleavage assay to investigate whether this mechanism operates for Fpg and OGG1, two structurally unrelated DNA glycosylases that excise an important oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), from DNA. Similar to a number of other DNA glycosylases or restriction endonucleases, Fpg and OGG1 processively excised 8-oxoG from pairs with cytosine at low salt concentrations, indicating that the lesion search likely proceeds by one-dimensional diffusion. At high salt concentrations, both enzymes switched to a distributive mode of lesion search. Correlated cleavage of abasic site-containing substrates proceeded in the same manner as cleavage of 8-oxoG. Interestingly, both Fpg and especially OGG1 demonstrated higher processivity if the substrate contained 8-oxoG.A pairs, against which these enzyme discriminate. Introduction of a nick into the substrate DNA did not decrease the extent of correlated cleavage, suggesting that the search probably involves hopping between adjacent positions on DNA rather than sliding along DNA. This was further supported by the observation that mutant forms of Fpg (Fpg-F110A and Fpg-F110W) with different sizes of the side chain of the amino acid residue inserted into DNA during scanning were both less processive than the wild-type enzyme. In conclusion, processive cleavage by Fpg and OGG1 does not correlate with their substrate specificity and under nearly physiological salt conditions may be replaced with the distributive mode of action.     

Characterization of a novel 8-oxoguanine-DNA glycosylase activity in Escherichia coli and identification of the enzyme as endonuclease VIII

8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.     

Poly(ADP-ribose) polymerase 1 (PARP-1) binds to 8-oxoguanine-DNA glycosylase (OGG1)

Human 8-oxoguanine-DNA glycosylase (OGG1) plays a major role in the base excision repair pathway by removing 8-oxoguanine base lesions generated by reactive oxygen species. Here we report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase 1 (PARP-1), a DNA-damage sensor protein involved in DNA repair and many other cellular processes. We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. Furthermore, OGG1 binds to PARP-1 through its BRCA1 C-terminal (BRCT) domain. OGG1 stimulated the poly(ADP-ribosyl)ation activity of PARP-1, whereas decreased poly(ADP-ribose) levels were observed in OGG1(-/-) cells compared with wild-type cells in response to DNA damage. Importantly, activated PARP-1 inhibits OGG1. Although the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, these proteins were defective in activating PARP-1. Furthermore, OGG1(-/-) cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.     

Ionic strength and magnesium affect the specificity of Escherichia coli and human 8-oxoguanine-DNA glycosylases

An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often directs the misincorporation of dAMP during replication. To prevent mutations, cells possess an enzymatic system for the removal of 8-oxoG. A key element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but not from 8-oxoG:A. We investigated the influence of various factors, including ionic strength, the presence of Mg(2+) and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite-base specificity of Escherichia coli Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A decreased sharply with increasing salt and Mg(2+) concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher opposite-base specificity when salt and Mg(2+) were at near-physiological concentrations. This tendency was observed with both Cl(-) and glutamate as the major anions in the reaction mixture. Kinetic and binding parameters for the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were determined under several different conditions. Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally. We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due to high ionic strength and Mg(2+).