Quantum-chemical investigation of tautomerization ways of Watson-Crick DNA base pair guanine-cytosine

A novel physico-chemical mechanism of the Watson-Crick DNA base pair Gua.Cyt tautomerization Gua.Cyt*<---->Gua.Cyt<---->Gua*.Cyt (mutagenic tautomers of bases are marked by asterisks) have been revealed and realized in a pathway of single proton transfer through two mutual isoenergetic transition states with Gibbs free energy of activation 30.4 and 30.6 kcal/mol and they are ion pairs stabilized by three (N2H...N3, N1H...N4- and O6+H...N4-) and five (N2H...O2, N1H...O2, N1H...N3, O6+H...N4- and 06+H...N4-) H-bonds accordingly. Stable base pairs Gua-Cyt* and Gua*.Cyt which dissociate comparably easy into monomers have acceptable relative Gibbs energies--12.9 and 14.3 kcal/mol--for the explanation of the nature of the spontaneous transitions of DNA replication. Results are obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-31 1++G(d,p) level of theory in vacuum approach.     

The physico-chemical “anatomy” of the tautomerization through the DPT of the biologically important pairs of hypoxanthine with DNA bases: QM and QTAIM perspectives

The biologically important tautomerization of the Hyp·Cyt, Hyp*·Thy and Hyp·Hyp base pairs to the Hyp*·Cyt*, Hyp·Thy* and Hyp*·Hyp* base pairs, respectively, by the double proton transfer (DPT) was comprehensively studied in vacuo and in the continuum with a low dielectric constant (ε?=?4) corresponding to hydrophobic interfaces of protein–nucleic acid interactions by combining theoretical investigations at the B3LYP/6-311++G(d,p) level of QM theory with QTAIM topological analysis. Based on the sweeps of the energetic, electron-topological, geometric and polar parameters, which describe the course of the tautomerization along the intrinsic reaction coordinate (IRC), it was proved that the tautomerization through the DPT is concerted and asynchronous process for the Hyp·Cyt and Hyp*·Thy base pairs, while concerted and synchronous for the Hyp·Hyp homodimer. The continuum with ε?=?4 does not affect qualitatively the course of the tautomerization reaction for all studied complexes. The nine key points along the IRC of the Hyp·Cyt?Hyp*·Cyt* and Hyp*·Thy?Hyp·Thy* tautomerizations and the six key points of the Hyp·Hyp?Hyp*·Hyp* tautomerization have been identified and fully characterized. These key points could be considered as electron-topological “fingerprints” of concerted asynchronous (for Hyp·Cyt and Hyp*·Thy) or synchronous (for Hyp·Hyp) tautomerization process via the DPT. It was found, that in the Hyp*·Cyt*, Hyp·Thy*, Hyp·Hyp and Hyp*·Hyp* base pairs all H-bonds are significantly cooperative and mutually reinforce each other, while the C2H…O2 H-bond in the Hyp·Cyt base pair and the O6H…O4 H-bond in the Hyp*·Thy base pair behave anti-cooperatively, i.e., they become weakened, while two others become strengthened.     

Base modifications in plasmid DNA caused by potassium permanganate

KMnO4 is a powerful oxidizing agent which has been used to modify DNA bases. In previous studies, mild KMnO4 treatment has been shown to preferentially modify Thy; Cyt and Gua are modified only under harsher conditions to as yet unidentified products. In the present study, denatured plasmid pCMV beta gal DNA was exposed to 0.015-1.5 mM KMnO4, pH 8.6, at 4 degrees C for 5 min, after which the DNA was hydrolyzed in formic acid, trimethylsilylated, and analyzed for modified base content by gas chromatography-mass spectrometry/selected ion monitoring. KMnO4 treatment, even at concentrations as low as 0.015 mM, caused a concentration-dependent increase in the Thy products Thy glycol and 5-hydroxy-5-methylhydantoin, the Cyt products Cyt glycol, 5,6-dihydroxycytosine, and 5-hydroxyhydantoin, the Ade product 8-hydroxyadenine, and the Gua product 8-hydroxyguanine. The Ade product 4,6-diamino-5-formamidopyrimidine and the Gua product 2,6-diamino-4-hydroxy-5-formamidopyrimidine were minimally (less than or equal to 2-fold) increased by treatment with greater than or equal to 0.8 mM KMnO4. These data demonstrate that, in addition to Thy, Cyt, Gua, and Ade bases in plasmid DNA may be modified by treatment with KMnO4, even under mild conditions. They represent the first identification of Cyt, Gua, and Ade products caused by KMnO4 treatment. Furthermore, these data suggest that previous studies which have used treatment with KMnO4 to study the mutagenicity of Thy glycol specifically or as a Thy-specific probe in DNA structure should be interpreted with caution.     

Theoretical study of the pre- and post-translational effects of adenine and thymine tautomers and methyl derivatives

The study of pre-translational effects (ionization, tautomerization) and post-translational effects (methylation) of adenine and thymine has only recently been the focus of some studies. These effects can potentially help regulate gene expression as well as potentially disrupt normal gene function. Because of this wide array of roles, greater insight into these effects in deoxyribonucleic acids (DNA) are paramount. There has been considerable research of each phenomenon (tautomerization, methylation and ionization) individually. In this work, we attempt to shed light upon the pre-translational effects and post translational effects of adenine and thymine by investigating the electron affinities (EAs) and ionization potentials (IPs) of the major and minor tautomers and their methyl derivatives. We performed all calculations using the density functional theory (DFT) B3LYP functional accompanied with 6-311G(d,p), 6-311+G(d,p) and 6-311++G(df,pd) basis sets. Our results reveal that the thymine tautomer has a higher EA and IP than the adenine tautomers. The higher EA suggests that an electron that attaches to the AT base pair would predominately attach to the thymine instead of adenine. The higher IP would suggest that an electron that is removed from the AT base pair would be predominately removed from the adenine within the base pair. Understanding how tautomerization, ionization and methylation differences change effects, discourages, or promotes one another is lacking. In this work, we begin the steps of integrating these effects with one another, to gain a greater understanding of molecular changes in DNA bases.     

The influence of Li+, Na+, Mg2+, Ca2+, and Zn2+ ions on the hydrogen bonds of the Watson-Crick base pairs

The interaction of mono- and divalent metal ions with the nucleic acid base pairs A:T and G:C has been studied using ab initio self-consistent field Hartree-Fock computations with minimal basis sets. Energy-optimized structures of the two base pairs with a final base-base distance of L = 10.35 A have been determined and were further used in calculations on ternary complexes Mn+ - A:B together with previously computed coordination geometries of the cations at adenine (Ade), thymine (Thy), and guanine (Gua). Besides the binding energy of the various metal ions to the base pairs, changes in the stability of the H bonds between Ade and Thy or Gua and Cyt have been determined. Polarization effects of the metal ion on the ligand turned out to increase the binding between complementary bases. Regardless of the metal species, cation binding to Gua N(3) and Thy O(2) leads to a special increase in H-bond stability, whereas binding to Ade N(3) changes the H-bond stability least. Situated in between are the stabilizing effects caused by Gua and Ade N(7) coordination. A remarkable relation between the stability of the H bond and the distance from metal binding site to H bonds was found. This relationship has been rationalized in terms of partial charges of the atoms participating in H bonding, which can reveal the trend in the electrostatic part of total H bond energy. It can be shown that a short distance between coordination site and acceptor hydrogen increases the H-bond strength substantially, while a long distance shows minor effects as supposed. On the other hand, the opposite effect is observed for the influence of the distance between binding site and donor atom. A comparison of our findings with a new model of transition metal ion facilitated rewinding of denatured DNA proposed by S. Miller, D. VanDerveer, and L. Marzilli is given [(1985) J. Am. Chem. Soc. 107, 1048-1055].     

Biomolecular response of oxanine in DNA strands to T4 polynucleotide kinase, T4 DNA ligase, and restriction enzymes

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO₂-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5′-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa > Gua > adenine (Ade) ∼ thymine (Thy) > cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.     

Structural effects of nucleobase variations at key active site residue Ade38 in the hairpin ribozyme

The hairpin ribozyme requires functional groups from Ade38 to achieve efficient bond cleavage or ligation. To identify molecular features that contribute to catalysis, structures of position 38 base variants 2,6-diaminopurine (DAP), 2-aminopurine (AP), cytosine (Cyt), and guanine (Gua) were determined between 2.2 and 2.8 A resolution. For each variant, two substrate modifications were compared: (1) a 2'-O-methyl-substituent at Ade-1 was used in lieu of the nucleophile to mimic the precatalytic state, and (2) a 3'-deoxy-2',5'-phosphodiester linkage between Ade-1 and Gua+1 was used to mimic a reaction-intermediate conformation. While the global fold of each variant remained intact, the results revealed the importance of Ade38 N1 and N6 groups. Absence of N6 resulting from AP38 coincided with failure to localize the precatalytic scissile phosphate. Cyt38 severely impaired catalysis in a prior study, and its structures here indicated an anti base conformation that sequesters the imino moiety from the scissile bond. Gua38 was shown to be even more deleterious to activity. Although the precatalytic structure was nominally affected, the reaction-intermediate conformation indicated a severe electrostatic clash between the Gua38 keto oxygen and the pro-Rp oxygen of the scissile bond. Overall, position 38 modifications solved in the presence of 2'-OMe Ade-1 deviated from in-line geometry, whereas variants with a 2',5' linkage exhibited S-turn destabilization, as well as base conformational changes from syn to anti. These findings demonstrate the importance of the Ade38 Watson-Crick face in attaining a reaction-intermediate state and the sensitivity of the RNA fold to restructuring when electrostatic and shape features fail to complement.     

The nature of the transition mismatches with Watson–Crick architecture: the G*·T or G·T* DNA base mispair or both? A QM/QTAIM perspective for the biological problem

This study provides the first accurate investigation of the tautomerization of the biologically important guanine*·thymine (G*·T) DNA base mispair with Watson–Crick geometry, involving the enol mutagenic tautomer of the G and the keto tautomer of the T, into the G·T* mispair (?G?=?.99?kcal?mol^?1, population?=?15.8% obtained at the MP2 level of quantum-mechanical theory in the continuum with ε?=?4), formed by the keto tautomer of the G and the enol mutagenic tautomer of the T base, using DFT and MP2 methods in vacuum and in the weakly polar medium (ε?=?4), characteristic for the hydrophobic interfaces of specific protein–nucleic acid interactions. We were first able to show that the G*·T?G·T* tautomerization occurs through the asynchronous concerted double proton transfer along two antiparallel O6H···O4 and N1···HN3 H-bonds and is assisted by the third N2H···O2 H-bond, that exists along the entire reaction pathway. The obtained results indicate that the G·T* base mispair is stable from the thermodynamic point of view complex, while it is dynamically unstable structure in vacuum and dynamically stable structure in the continuum with ε?=?4 with lifetime of 6.4·10^?12?s, that, on the one side, makes it possible to develop all six low-frequency intermolecular vibrations, but, on the other side, it is by three orders less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. One of the more significant findings to emerge from this study is that the short-lived G·T* base mispair, which electronic interaction energy between the bases (?23.76?kcal?mol^?1) exceeds the analogical value for the G·C Watson–Crick nucleobase pair (?20.38?kcal?mol^?1), “escapes from the hands” of the DNA replication machinery by fast transforming into the G*·T mismatch playing an indirect role of its supplier during the DNA replication. So, exactly the G*·T mismatch was established to play the crucial role in the spontaneous point mutagenesis.     

Whether 2-aminopurine induces incorporation errors at the DNA replication? A quantum-mechanical answer on the actual biological issue

In this paper, we consider the mutagenic properties of the 2-aminopurine (2AP), which has intrigued molecular biologists, biophysicists and physical chemists for a long time and been widely studied by both experimentalists and theorists. We have shown for the first time using QM calculations, that 2AP very effectively produces incorporation errors binding with cytosine (C) into the wobble (w) C·2AP(w) mispair, which is supported by the N4H?N1 and N2H?N3 H-bonds and is tautomerized into the Watson–Crick (WC)-like base mispair C*·2AP(WC) (asterisk denotes the mutagenic tautomer of the base), that quite easily in the process of the thermal fluctuations acquires enzymatically competent conformation. 2AP less effectively produces transversions forming the wobble mispair with A base – A·2AP(w), stabilized by the participation of the N6H?N1 and N2H?N1 H-bonds, followed by further tautomerization A·2AP(w) → A*·2AP(WC) and subsequent conformational transition A*·2AP(WC) → A*·2AP_syn thus acquiring enzymatically competent structure. In this case, incorporation errors occur only in those case, when 2AP belongs to the incoming nucleotide. Thus, answering the question posed in the title of the article, we affirm for certain that 2AP induces incorporation errors at the DNA replication. Obtained results are consistent well with numerous experimental data.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

Reactions of 4‐[Bis(2‐chloroethyl)amino]benzenebutanoic Acid (Chlorambucil) with DNA

4‐[Bis(2‐chloroethyl)amino]benzenebutanoic acid (=chlorambucil, 1 ; 2.5 mM ) was allowed to react with single‐ and double‐stranded calf thymus DNA at physiological pH (cacodylic acid, 50% base) at 37°. The DNA–chlorambucil adducts were identified by analyzing the DNA hydrolysates by NMR, UV, HPLC, LC/ESI‐MS/MS techniques as well as by spiking with authentic materials. ssDNA was more reactive than dsDNA, and the order of reactivity in ssDNA was Ade‐N1>Gua‐N7>Cyt‐N3>Ade‐N3. The most reactive site in dsDNA was Ade‐N3. The Gua‐N7 and Ade‐N3 adducts were hydrolytically labile. Ade‐N7 adduct could not be identified in the hydrolysates of ssDNA or dsDNA. The adduct Gua‐N7,N7, which consists of two units of Gua bound together with a unit derived from chlorambucil, is a cross‐linking adduct, and it was detected in the hydrolysates of ssDNA and dsDNA. Also several other adducts were detected which could be characterized by spiking with previously isolated authentic adducts or tentatively by MS. The role of chlorambucil–DNA adducts on the cytotoxicity and mutagenity of 1 is also discussed.     

Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.     

Experimental and theoretical study of energetics of complex formation between nucleic acid bases and the bases with amide group.

A number of nucleic acid base pairs and complexes between the bases and the amide group of acrylamide have been studied experimentally by using mass spectrometry and theoretically by the method of atom-atom potential function calculations. It has been found from temperature dependencies of peak intensities in mass spectra of m2.2.9(3) Gua.m1Ura, m9 Ade.m1Cyt, m2.2.9(3) Gua.m1Gua.m1Cyt pairs that enthalpy values, delta H, of the complex formation are equal to 14.2 +/- 1.1, 13.5 +/- 1.3 and 16.4 +/- 1.4 kcal/M, respectively, and those of acrylamide with m1.3(2) Ura and m1Thy corresponds to 9.7 +/- 1.0 and 6.8 +/- 0.6 kcal/M. There is a good agreement of the experimental data with calculations when taking into account both the amino-oxo and the amino-hydroxy tautomeric forms of guanine. A combined use of the data allows us to determine the energy, the modes of interaction and the structure of the complexes. The results are discussed in connection with the modelling of molecular structure of biopolymers by the method of classical potential functions, protein-nucleic acids recognition and fidelity of nucleic acids biosynthesis.     

Molecular models of neocarzinostatin damage of DNA: analysis of sequence dependence in 5''GAGCG:5''CGCTC.

Model building and molecular mechanics and dynamics calculations have been performed on a number of complexes of the post-activated form of the neocarzinostatin chromophore (NCS) with the B-DNA oligomer 5'GAGCG:5'CGCTC. Stable structures with the naphthoic acid moiety intercalated at all base pairs can be constructed. The observed bistranded lesions consisting of an abasic site at the Cyt residue in AGC and a direct break at the Thy residue on the complementary strand can be explained by assuming that NCS in the (R,R) form intercalates between the Ade2-Thy9/Gua3-Cyt8 base step with its 'diradical' core oriented towards the 3'-end of the (+) strand. Sites at C5', C4' and C1' in the minor groove are within a short enough distance from the two radical centers on NCS to permit hydrogen atom abstraction and the formation of the bistranded lesions. Strand cleavage at Thy9 may occur as a single lesion if NCS is intercalated into the Gua3-Cyt8/Cyt4-Gua7 base step with its active core towards the 3'-end of the (-) strand. The results are analyzed, and the utility and limitations of this type of model building are discussed.     

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

A recently solved Dnmt1-DNA crystal structure revealed several enzyme-DNA contacts and large structural rearrangements of the DNA at the target site, including the flipping of the non-target strand base of the base pair flanking the CpG site and formation of a non-canonical base pair between the non-target strand Gua and the flanking base pair. Here, we show that the contacts of the enzyme to the target base and the Gua:5mC base pair that are observed in the structure are very important for catalytic activity. The contacts to the non-target strand Gua are not important since its exchange by Ade stimulated activity. Except target base flipping, we could not find evidence that the DNA rearrangements have a functional role.     

Slow loss of deoxyribose from the N7deoxyguanosine adducts of estradiol-3,4-quinone and hexestrol-3',4'-quinone. Implications for mutagenic activity

A variety of evidence has been obtained that estrogens are weak tumor initiators. A major step in the multi-stage process leading to tumor initiation involves metabolic formation of 4-catechol estrogens from estradiol (E2) and/or estrone and further oxidation of the catechol estrogens to the corresponding catechol estrogen quinones. The electrophilic catechol quinones react with DNA mostly at the N-3 of adenine (Ade) and N-7 of guanine (Gua) by 1,4-Michael addition to form depurinating adducts. The N3Ade adducts depurinate instantaneously, whereas the N7Gua adducts depurinate with a half-life of several hours. Only the apurinic sites generated in the DNA by the rapidly depurinating N3Ade adducts appear to produce mutations by error-prone repair. Analogously to the catechol estrogen-3,4-quinones, the synthetic nonsteroidal estrogen hexestrol-3',4'-quinone (HES-3',4'-Q) reacts with DNA at the N-3 of Ade and N-7 of Gua to form depurinating adducts. We report here an additional similarity between the natural estrogen E2 and the synthetic estrogen HES, namely, the slow loss of deoxyribose from the N7deoxyguanosine (N7dG) adducts formed by reaction of E2-3,4-Q or HES-3',4'-Q with dG. The half-life of the loss of deoxyribose from the N7dG adducts to form the corresponding 4-OHE2-1-N7Gua and 3'-OH-HES-6'-N7Gua is 6 or 8 h, respectively. The slow cleavage of this glycosyl bond in DNA seems to limit the ability of these adducts to induce mutations.     

Carbenic vs. ionic mechanistic pathway in reaction of cyclohexanone with bromoform

The extensive computation study was done to elucidate the mechanism of formation dibromoepoxide from cyclohexanone and bromoform. In this reaction, the formation of dihaloepoxide 2 is postulated as a key step that determines the distribution and stereochemistry of products. Two mechanistic paths of reaction were investigated: the addition of dibromocarbene to carbonyl group of ketone, and the addition of tribromomethyl carbanion to the same (C=O) group. The mechanisms for the addition reactions of dibromocarbenes and tribromomethyl carbanions with cyclohexanone have been investigated using ab initio HF/6-311++G** and MP2/6-311+G* level of theory. Solvent effects on these reactions have been explored by calculations which included a continuum polarizable conductor model (CPCM) for the solvent (H(2)O). The calculations showed that both mechanisms are possible and are exothermic, but have markedly different activation energies.     

Quantum-chemical investigation of the elementary molecular mechanisms of pyrimidine-purine transversions

Purine-purine mispairs of DNA (thus involving template base in anti-conformation along the glycosidic bond and base of the incoming nucleotide - in syn-conformation) leading to pyrimidine-purine "transversions"-type point mutations were revealed and characterized at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory in vacuum approach adequately modeling hydrophobic environment of the active centre of high-fidelity replicative DNA-polymerases.     

Biophysical stability and enzymatic recognition of oxanine in DNA

Oxanine (Oxa), which is one of the major products generated from guanine by nitrosative oxidation and is as long-lived as Gua in DNA, has been thought to be one of the major causes for NO-induced DNA damage. In the present study, using several synthetic Oxa-containing oligodeoxynucleotides, biophysical stability and enzymatic recognition of Oxa was investigated in DNA strands. It was found that Oxa did not mediate marked distortion in the whole DNA structure although Oxa pairing with 4 normal bases decreased thermal stability of the DNA duplexes compared to Gua:Cyt base pair. Regarding the responses of the DNA-relevant enzymes to Oxa, it was determined that Oxa was recognized as Gua except that DNA polymerases incorporated Thy as well as Cyt opposite Oxa. These results imply that Oxa tends to behave as a kind of naturally occurring base, Gua and therefore, would be involved in the genotoxic and cytotoxic threats of NO in cellular system.