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.     

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.     

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.     

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.     

High resolution characterization of formamidopyrimidine-DNA glycosylase interaction with its substrate by chemical cross-linking and mass spectrometry using substrate analogs

Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOgg1) initiate the base excision repair pathway for 7,8-dihydro-8-oxoguanine (8-oxoG) residues present in DNA. Recent structural and biochemical studies of Fpg-DNA and hOgg1-DNA complexes point to the existence of extensive interactions between phosphate groups and amino acids. However, the role of these contacts and their physiological relevance remains unclear. In the present study, we combined chemical cross-linking and electrospray ionization mass spectrometry (ESI/MS/MS) approaches to identify interacting residues in the Fpg-DNA and hOgg1-DNA complexes. The active centers of Fpg and hOgg1 were cross-linked with a series of reactive oligonucleotide duplexes containing both a single 8-oxoG residue and an O-ethyl-substituted pyrophosphate internucleotide (SPI) group at different positions in duplex DNA. The cross-linking efficiency reached 50% for Fpg and 30% for hOgg1. We have identified seven phosphate groups on both strands of the DNA duplex specifically interacting with nucleophilic amino acids in Fpg, and eight in hOgg1. MS/MS analysis of the purified proteolytic fragments suggests that lysine 56 of Fpg and lysine 249 of hOgg1 cross-link to the phosphate located 3' to the 8-oxoG residue. Site-specific mutagenesis analysis of Fpg binding to DNA substrate confirms the conclusions of our approach. Our results are consistent with crystallographic data on the Fpg-DNA complex and provide new data on the hOgg1-DNA interaction. The approach developed in this work provides a useful tool to study pro- and eukaryotic homologues of Fpg as well as 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%.     

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.     

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site.To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.     

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%.     

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.     

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.

     

Thermodynamic,kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli

X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.     

Stepwise translocation of Dpo4 polymerase during error-free bypass of an oxoG lesion

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG•C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain–DNA phosphate contacts translocate by one nucleotide step, while the thumb domain–DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain–phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.     

Thermodynamics of the multi-stage DNA lesion recognition and repair by formamidopyrimidine-DNA glycosylase using pyrrolocytosine fluorescence--stopped-flow pre-steady-state kinetics

Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8-oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10-30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van't Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.     

Single-stranded oligodeoxyribonucleotides are substrates of Fpg protein from Escherichia coli

The interaction of Escherichia coli Fpg protein, which catalyzes excision of several damaged purine bases including 8-oxoguanine (oxoG) from DNA with a set of single- (ss) and double-stranded (ds) 23-mer oligodeoxyribonucleotides (ODNs) containing 8-oxoguanine(s) at various positions, has been investigated. The affinities of different ss ODNs (KM = 0.55-1.3 microM) were shown to be 12-170 times less than those for corresponding ds ODNs (KM = 6-60 nM). Depending on the position of the oxoG within the ODNs, relative initial rates of conversion of ss substrates may be less than, comparable, or greater than those for ds ODNs. The enzyme can remove 5'-terminal oxoG from ODNs only if the 5'-end is phosphorylated. Fpg does not release oxoG residues from the ultimate and penultimate 3'-terminal positions. Duplexes containing two adjacent oxoG are poor substrates for the glycosylase.     

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.     

Site-directed modification of DNA duplexes by chemical ligation.

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.     

Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.     

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.