Interactions of pro- and eukaryotic DNA repair enzymes with oligodeoxyribonucleotides containing clustered lesions

Interactions of oxoGuanine-DNA glycosylases from Escherichia coli (Fpg) and human (OGG1) and abasic site endonucleases from yeast (Apnl) and E. coli (Nfo) with oligodeoxyribonucleotides containing oxoGuanine (oxoG) and tetrahydrofuran (F, a stable analog of an abasic site) separated by various numbers of nucleotides have been studied. Inhibitor analysis has shown that the affinity of Fpg for single-stranded ligands does not depend on the relative positions of oxoG and F lesions. KM and kcat values have been determined for all double-stranded substrates and all enzymes under study. The effect of introducing the second lesion was strongly dependent on the relative positions of the lesions and the nature of the enzyme. The highest drop in the affinity (1.6-148-fold) and the reaction rate (4.8-58-fold) has been observed with Fpg and OGG1 for substrates containing F immediately 5' or 3' adjacent to oxoG. Introduction of the second lesion barely changed the KM values for Apnl and Nfo substrates. At the same time, the reaction rates were 5-10-fold lower for substrates containing two adjacent lesions. For all enzymes studied, increasing the distance between two lesions in duplex DNA reduced the effect of the lesion in KM and kcat values.     

Structural requirements of double and single stranded DNA substrates and inhibitors, including a photoaffinity label, of Fpg protein from Escherichia coli

Fpg protein (formamidopyrimidine or 8-oxoguanine DNA glycosylase) from E. coli catalyzes excision of several damaged purine bases, including 8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine from DNA. In this study the interaction of E. coli Fpg with various specific and nonspecific oligodeoxynucleotides was analyzed. Fpg was shown to remove 8-oxoguanine efficiently, not only from double-stranded, but also from single-stranded oligodeoxynucleotides. The Michaelis constants (KM) of a range of single-stranded oligodeoxynucleotides (0.55-1.3 microM) were shown to be 12-170 times higher that those for corresponding double-stranded oligodeoxynucleotides (KM = 6-60 nM). Depending on the position of the 8-oxoguanine within the oligodeoxynucleotides, relative initial rates of conversion of single-stranded substrates were found to be lower than, comparable to, or higher than those for double-stranded oligodeoxynucleotides. The enzyme can interact effectively not only with specific, but also with nonspecific single-stranded and double-stranded oligodeoxynucleotides, which are competitive inhibitors of the enzyme towards substrate. Fpg became irreversibly labeled after UV-irradiation in the presence of photoreactive analogs of single-stranded and double-stranded oligodeoxynucleotides. Specific and nonspecific single-stranded and double-stranded oligodeoxynucleotides essentially completely prevented the covalent binding of Fpg by the photoreactive analog. All these data argue for similar interactions occurring in the DNA binding cleft of the enzyme with both specific and nonspecific oligodeoxynucleotides. The relative affinities of Fpg for specific and nonspecific oligodeoxynucleotides differ by no more than 2 orders of magnitude. Addition of the second complementary chain increases the affinity of the first single-stranded chain by a factor of approximately 10. It is concluded that Michaelis complex formation of Fpg with DNA containing 8-oxoG cannot alone provide the major part of the enzyme specificity, which is found to lie in the kcat term for catalysis; the reaction rate being increased by 6-7 orders of magnitude by the transition from nonspecific to specific oligodeoxynucleotides.     

Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups.

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.     

Cross-linking of Escherichia coli formamidopyrymidine-DNA glycosylase to DNA duplexes containing photoactivatable phenyl(trifluoromethyl)diazirine groups

New reactive analogs of substrates for DNA repair enzyme E. coli Fpg protein containing the residues of 8-oxoguanine and photoactivatable phenyl(trifluoromethyl)diazirine groups were synthesized. Their substrate properties were investigated. Using photocross-linking technique, we established the presence of contacts of two nucleosides located near the oxoG with amino acids from the Fpg protein. The cross-linking efficiency achieved 10%.     

Cross-Linking of Escherichia coli Formamidopyrymidine-DNA Glycosylase to DNA Duplexes Containing Photoactivatable Phenyl(Trifluoromethyl)diazirine Groups

Abstract

New reactive analogs of substrates for DNA repair enzyme E. coli Fpg protein containing the residues of 8-oxoguanine and photoactivatable phenyl(trifluoromethyl)diazirine groups were synthesized. Their substrate properties were investigated. Using photocross-linking technique, we established the presence of contacts of two nucleosides located near the oxoG with amino acids from the Fpg protein. The cross-linking efficiency achieved 10%.     

New non-hydrolyzable substrate analogs for 8-oxoguanine-DNA glycosylases

8-Oxoguanine-DNA glycosylases play a key role in the repair of oxidatively damaged DNA. The Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are DNA base excision repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (oxoG) residue, and cleave DNA strand. Specific contacts between DNA phosphate groups and amino acids from active centers of these enzymes play a significant role in DNA-protein interactions. In order to design new non-hydrolyzable substrate analogs of Fpg and hOGG1 for structural studies modified DNA duplexes containing pyrophosphate or OEt-substituted pyrophosphate internucleotide (SPI) groups near the damage were tested. We showed that enzymes recognize and specifically bind to DNA duplexes obtained. The mechanism of incision of oxoG by the Fpg and hOGG1 was determined. We revealed that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the oxoG. In contrast, Fpg and hOGG1 effectively incise DNA duplex carrying analogous phosphate modifications 5' to the oxoG. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or substituted pyrophosphate groups immediately 3' to the oxoG are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a oxoG-containing DNA bound to catalytically active wild-type enzymes as well as their pro- and eucaryotic homologs.     

Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.     

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.     

DNA lesion recognition by the bacterial repair enzyme MutM

MutM is a bacterial DNA glycosylase that removes the mutagenic lesion 8-oxoguanine (oxoG) from duplex DNA. The means of oxoG recognition by MutM (also known as Fpg) is of fundamental interest, in light of the vast excess of normal guanine bases present in genomic DNA. The crystal structure of a recognition-competent but catalytically inactive version of MutM in complex with oxoG-containing DNA reveals the structural basis for recognition. MutM binds the oxoG nucleoside in the syn glycosidic configuration and distinguishes oxoG from guanine by reading out the protonation state of the N7 atom. The segment of MutM principally responsible for oxoG recognition is a flexible loop, suggesting that conformational mobility influences lesion recognition and catalysis. Furthermore, the structure of MutM in complex with DNA containing an alternative substrate, dihydrouracil, demonstrates how MutM is able to recognize lesions other than oxoG.     

The influence of formamidopyrimidine-DNA glycosylase on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZ alpha gene.

Base excision repair (BER) is a very important repair mechanism to cope with oxidative DNA damage. One of the most predominating oxidative DNA damages after exposure to ionizing radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This damage is repaired by formamidopyrimidine-DNA glycosylase (Fpg), a DNA glycosylase which is part of BER. Correct repair of 8oxoG is of great importance for cells, because 8oxoG has strong miscoding properties. Mispairing of 8oxoG with adenine instead of cytosine results in G:C to T:A transversion mutations. To determine the effect of a Fpg-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene, double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target, was irradiated with gamma-rays in aqueous solution under oxic conditions. Subsequently, the DNA was transfected into a wild-type Escherichia coli strain (JM105) and an isogenic Fpg-deficient E. coli strain (BH410). Although the overall spontaneous mutation spectra between JM105 and BH410 seemed similar, remarkable differences could be observed when the individual base pair substitutions were viewed. The amount of C to A transversions, which are most probably caused by unrepaired 8oxoG, has increased 3. 5-fold in the spontaneous BH410 spectrum. When the gamma-radiation-induced mutation spectra of JM105 and BH410 were compared, there was even a larger increase of C to A transversions in the BH410 strain (7-fold). We can therefore conclude that the straightforward approach used in this study confirms the importance of Fpg in repair of gamma-radiation-induced damage, and most probably especially in the repair of 8oxoG.     

The influence of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZalpha gene of m13mp10

One of the most predominating oxidative DNA damages, both spontaneously formed and after gamma-radiation is 7, 8-dihydro-8-oxoguanine (8oxoG). This 8oxoG is a mutagenic lesion because it can mispair with adenine instead of the correct cytosine leading to G:C to T:A transversions. In Escherichia coli (E. Coli) base excision repair (BER) is one of the most important repair systems for the repair of 8oxoG and other oxidative DNA damage. An important part of BER in E. coli is the so-called GO system which consists of three repair enzymes, MutM (Fpg), MutY and MutT which are all involved in repair of 8oxoG or 8oxoG mispairs. The aim of this study is to determine the effect of combined Fpg- and MutY-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum of the lacZalpha gene. For that purpose, non-irradiated or gamma-irradiated double-stranded (ds) M13mp10 DNA, with the lacZalpha gene inserted as mutational target sequence was transfected into an E. coli strain which is deficient in both Fpg and MutY (BH1040). The resulting mutation spectra were compared with the mutation spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105) which were determined in an earlier study. The results of the present study indicate that combined Fpg- and MutY-deficiency induces a large increase in G:C to T:A transversions in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)) as compared to the fpg(-) and the wild type strain. Besides the increased levels of G:C to T:A transversions, there is also an increase in G:C to C:G transversions and frameshift mutations in both the spontaneous and gamma-radiation-induced mutation spectra of BH1040 (fpg(-)mutY(-)).     

New Noncleavable Analogs of 8-Oxoguanine-DNA Glycosylase Substrates

8-Oxoguanine-DNA glycosylases play a key role in repairing oxidatively damaged DNA. Excision repair enzymes Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein) and human 8-oxoguanine-DNA glycosylase (hOGG1) catalyze excision of 7,8-dihydro-8-oxoguanine (oxoG) from DNA and subsequent cleavage of the sugar–phosphate backbone. Contacts between DNA phosphate groups and amino acid residues of the active centers of the enzymes are of importance for specific binding and catalysis. To construct noncleavable analogs of Fpg protein and hOGG1 substrates, modifications of phosphate groups bound to a damaged nucleotide were tested for their effect on the substrate properties of modified DNA duplexes. New oxoG-containing analogs of Fpg protein and hOGG1 substrates were synthetic DNA duplexes that contained a pyrophosphate or a substituted pyrophosphate group bound with the 5- or 3-OH of 8-oxoguanosine. The duplexes proved to be recognized and specifically bound by Fpg protein and hOGG1. Analysis of the mechanism of their interaction with Fpg protein and hOGG1 showed that modification of the internucleotide phosphate bound with 3-OH of 8-oxoguanosine prevents oxoG excision from DNA. Yet both enzymes efficiently cleaved the DNA duplexes when the modified phosphate was bound with the 5-OH of 8-oxoguanosine. DNA duplexes with a pyrophosphate or substituted pyrophosphate group at 3-OH of 8-oxoguanosine are noncleavable analogs of 8-oxoguanine-DNA glycosylase substrates and can be used to study the structures of catalytically active forms of Fpg protein and hOGG1 and their prokaryotic or eukaryotic homologs in complex with oxoG-containing DNA.     

Molecular dynamics simulation of the opposite-base preference and interactions in the active site of formamidopyrimidine-DNA glycosylase

Background

Formamidopyrimidine-DNA glycosylase (Fpg) removes abundant pre-mutagenic 8-oxoguanine (oxoG) bases from DNA through nucleophilic attack of its N-terminal proline at C1′ of the damaged nucleotide. Since oxoG efficiently pairs with both C and A, Fpg must excise oxoG from pairs with C but not with A, otherwise a mutation occurs. The crystal structures of several Fpg–DNA complexes have been solved, yet no structure with A opposite the lesion is available.

Results

Here we use molecular dynamic simulation to model interactions in the pre-catalytic complex of Lactococcus lactis Fpg with DNA containing oxoG opposite C or A, the latter in either syn or anti conformation. The catalytic dyad, Pro1–Glu2, was modeled in all four possible protonation states. Only one transition was observed in the experimental reaction rate pH dependence plots, and Glu2 kept the same set of interactions regardless of its protonation state, suggesting that it does not limit the reaction rate. The adenine base opposite oxoG was highly distorting for the adjacent nucleotides: in the more stable syn models it formed non-canonical bonds with out-of-register nucleotides in both the damaged and the complementary strand, whereas in the anti models the adenine either formed non-canonical bonds or was expelled into the major groove. The side chains of Arg109 and Phe111 that Fpg inserts into DNA to maintain its kinked conformation tended to withdraw from their positions if A was opposite to the lesion. The region showing the largest differences in the dynamics between oxoG:C and oxoG:A substrates was unexpectedly remote from the active site, located near the linker joining the two domains of Fpg. This region was also highly conserved among 124 analyzed Fpg sequences. Three sites trapping water molecules through multiple bonds were identified on the protein–DNA interface, apparently helping to maintain enzyme-induced DNA distortion and participating in oxoG recognition.

Conclusion

Overall, the discrimination against A opposite to the lesion seems to be due to incorrect DNA distortion around the lesion-containing base pair and, possibly, to gross movement of protein domains connected by the linker.

     

DNA damage by the sulfate radical anion: hydrogen abstraction from the sugar moiety versus one-electron oxidation of guanine

The products of oxidative damage to double-stranded (ds) DNA initiated by photolytically generated sulfate radical anions SO₄^?? were analyzed using reverse-phase (RP) high-performance liquid chromatography (HPLC). Relative efficiencies of two major pathways were compared: production of 8-oxoguanine (8oxoG) and hydrogen abstraction from the DNA 2-deoxyribose moiety (dR) at C1,′ C4,′ and C5′ positions. The formation of 8oxoG was found to account for 87% of all quantified lesions at low illumination doses. The concentration of 8oxoG quickly reaches a steady state at about one 8oxoG per 100 base pairs due to further oxidation of its products. It was found that another guanine oxidation product identified as 2-amino-5-(2′-alkylamino)-4H-imidazol-4-one (X) was released in significant quantities from its tentative precursor 2-amino-5-[(2′-deoxy-β-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) upon treatment with primary amines in neutral solutions. The linear dose dependence of X release points to the formation of dIz directly from guanine and not through oxidation of 8oxoG. The damage to dR was found to account for about 13% of the total damage, with majority of lesions (33%) originating from the C4′ oxidation. The contribution of C1′ oxidation also turned out to be significant (17% of all dR damages) despite of the steric problems associated with the abstraction of the C1′-hydrogen. However, no evidence of base-to-sugar free valence transfer as a possible alternative to direct hydrogen abstraction at C1′ was found.     

A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine–DNA glycosylase

In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme–DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson–Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes.     

The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene

Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage. A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages. Examples of DNA damages which are recognized by NER are thymine glycol and abasic sites (AP sites). The main objective of this study is to determine if NER can act as a backup system for the repair of spontaneous and gamma-radiation-induced damages when Fpg is deficient. For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105), which were determined in an earlier study. Furthermore, to examine effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E. coli strain in which only UvrA is deficient (BH430) were also determined and compared with a wild type E. coli strain (JM105). The results of this study indicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions. The gamma-radiation-induced mutation spectrum of BH420 (fpg(-)uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutations, as compared to BH410 where only Fpg is deficient. Based on these results, we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient. Instead, NER seems to make mistakes, leading to the formation of mutations.     

Interaction of pro-and eukaryotic DNA repair enzymes with oligodeoxyribonucleotides containing clustered lesions

A study was made of the interaction of 8-oxoguanine-DNA glycosylases of Escherichia coli (Fpg) and human (OGG1), as well as apurinic/apyrimidinic endonucleases of yeast (Apn1) and E. coli (Nfo), with oligodeoxyribonucleotides containing 8-oxoguaine (oxoG) and tetrahydrofuran (F, a stable analog of an apurinic site) separated by various numbers of nucleotides. Inhibitor analysis showed that the affinity of Fgp for single-stranded DNA ligands is virtually independent of the relative positions of oxoG and F. K _M and k _cat were determined for all the four enzymes and all double-stranded substrates studied. The effect of the second lesion strongly depended both on the relative position of the lesion and the enzyme of interest. The highest drop in the affinity of Fpg and OGG1 for the substrate (1.6-to 148-fold) and in the reaction rate (4.8-to 58-fold) was recorded for the oligonucleotides in which F was immediately 3′ or 5′ of oxoG. Introduction of the second lesion barely affected K _M for nucleases Apn1 and Nfo. The reaction rate was five-to tenfold lower for the substrates containing two adjacent lesions. For all enzymes studied, an increase in the distance between two lesions in double-stranded DNA decreased their contribution to K _M and k _cat.     

Use of crosslinking for revealing the DNA phosphate groups forming specific contacts with the E. coli Fpg protein

Specific contacts between DNA phosphate groups and positively charged nucleophilic amino acids from the Escherichia coli Fpg protein play a significant role in DNA-Fpg protein interaction. In order to identify these phosphate groups the chemical crosslinking procedure was carried out. The probing of the Fpg protein active center was performed using a series of reactive DNA duplexes containing both a single 7,8-dihydro-8-oxoguanosine (oxoG) residue and O-alkyl-substituted pyrophosphate internucleotide groups at the same time. Reactive internucleotide groups were introduced in dsDNA immediately 5' or 3' to the oxidative lesion and one or two nucleotides 5' or 3' away from it. We showed that the Fpg protein specifically binds to the modified DNA duplexes. The binding efficiency varied with the position of the reactive group and was higher for the duplexes containing substituted pyrophosphate groups at the ends of pentanucleotide with the oxoG in the center. The nicking efficiency of the DNA duplexes containing the reactive groups one or two nucleotides 5' away from the lesion was higher as compared to non-modified DNA duplex bearing only the oxidative damage. We found two novel non-hydrolizable substrate analogs for the Fpg protein containing pyrophosphate and substituted pyrophosphate groups 3' adjacent to the oxoG. Using crosslinking, we revealed the phosphate groups, 3' and 5' adjacent to the lesion, which have specific contacts with nucleophilic amino acids from the E. coli Fpg protein active center. The crosslinking efficiency achieved 30%. The approaches developed can be employed in the studies of pro- and eucaryotic homologs of the E. coli Fpg protein as well as other repair enzymes.     

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.     

Enzymatic properties of Escherichia coli and human 7,8-dihydro-8-oxoguanine DNA glycosylases.

Oxidative damage to DNA generates aberrant guanine bases such as 2,6-diamino-4-hydroxy-formamido-pyrimidine (Fapy) and 7,8-dihydro-8-oxoguanine (8-oxoG). Although synthetic oligonucleotides containing a single 8-oxoG have been widely used to study enzymatic processing of this lesion, the synthesis of oligonucleotides containing Fapy as a unique lesion has not been achieved to date. In this study, an oligonucleotide containing a single 2,6-diamino-4-hydroxy-5-(N-methyl)formamido-pyrimidine (me-Fapy, a methylated derivative of Fapy) was prepared by a DNA polymerase reaction and the subsequent alkali treatment. The repair activity of Fpg and hOGG1 proteins were compared using oligonucleotide substrates containing me-Fapy and 8-oxoG.