Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5'-d(…G*pT*…) and 5'-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.     

Oxidative DNA damage associated with combination of guanine and superoxide radicals and repair mechanisms via radical trapping

In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.     

Parallel-stranded DNA with Hoogsteen base pairing stabilized by a trans-[Pt(NH3)2]2+ cross-link: characterization and conversion into a homodimer and a triplex

The oligonucleotides 5'-d(TTTTCTTTTG) and 5'-d(AAAAGAAAAG) were cross-linked with a trans-[Pt(NH3)2]2+ entity via the N7 positions of the 3'-end guanine bases to give parallel-stranded (ps) DNA. At pH 4.2, CD and NMR spectroscopy indicate the presence of Hoogsteen base pairing. In addition, temperature-dependent UV spectroscopy shows an increase in melting temperature for the platinated duplex (35 degrees C) as compared to the non-platinated, antiparallel-stranded duplex formed from the same oligonucleotides (21 degrees C). A monomer-dimer equilibrium for the platinated 20mer is revealed by gel electrophoresis. At pH 4.2, addition of a third strand of composition 5'-d(AGCTTTTCTTTTAG) to the ps duplex leads to the formation of a triple helix with two distinct melting points at 38 degrees C (platinum cross-linked Hoogsteen part) and 21 degrees C (Watson-Crick part), respectively.     

Combination reactions of superoxide with 8-Oxo-7,8-dihydroguanine radicals in DNA: kinetics and end products

One of the major biomarkers of oxidative stress and oxidative damage of cellular DNA is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is more easily oxidized than guanine to diverse oxidative products. In this work, we have investigated further oxidative transformations of 8-oxoGua in single- and double-stranded oligonucleotides to the dehydroguanidinohydantoin, oxaluric acid, and diastereomeric spiroiminodihydantoin lesions. The relative distributions of these end products were explored by a combined kinetic laser spectroscopy and mass spectrometry approach and are shown to depend markedly on the presence of superoxide radical anions. The 8-oxaGua radicals were produced by one-electron oxidation of 8-oxoGua by 2-aminopurine radicals generated by the two-photon ionization of 2-aminopurine residues site specifically positioned in 5'-d(CC[2-aminopurine]TC[8-oxoGua]CTACC). The hydrated electrons also formed in the photoionization process were trapped by dissolved molecular oxygen thus producing superoxide. A combination reaction between the 8-oxoGua and superoxide radicals occurs with the rate constant of (1.3 +/- 0.2) x 10(8) m(-1) s(-1) and (1.0 +/- 0.5) x 10(8) m(-1) s(-1) in single- and double-stranded DNA, respectively. The major end products of this reaction are the dehydroguanidinohydantoin lesions that slowly hydrolyze to oxaluric acid residues. In the presence of Cu,Zn-superoxide dismutase, an enzyme that induces the rapid catalytic dismutation of superoxide to the less reactive H(2)O(2) and O(2), the yields of the dehydroguanidinohydantion lesions become negligible. Under these conditions, the 8-oxoGua radicals do not exhibit any observable reactivities with oxygen (k < 10(2) m(-1) s(-1)), decay on the time interval of several seconds, and the major reaction products are the spiroiminodihydantoin lesions. The possible biological implications of the 8-oxoGua oxidation are discussed.     

Generation of 5-(2'-deoxycytidyl)methyl radical and the formation of intrastrand cross-link lesions in oligodeoxyribonucleotides

Hydroxyl radical is one of the major reactive oxygen species (ROS) formed from γ-radiolysis of water or Fenton reaction, and it can abstract one hydrogen atom from the methyl carbon atom of thymine and 5-methylcytosine to give the 5-methyl radical of the pyrimidine bases. The latter radical can also be induced from Type-I photo-oxidation process. Here, we examined the reactivity of the independently generated 5-(2′-deoxycytidyl)methyl radical (I) in single- and double-stranded oligodeoxyribonucleotides (ODNs). It was found that an intrastrand cross-link lesion, in which the methyl carbon atom of 5-methylcytosine and the C8 carbon atom of guanine are covalently bonded, could be formed from the independently generated radical at both GmC and mCG sites, with the yield being much higher at the former site. We also showed by LC-MS/MS that the same cross-link lesions were formed in mC-containing duplex ODNs upon γ irradiation under both aerobic and anaerobic conditions, and the yield was ~10-fold higher under the latter conditions. The independently generated radical allows for the availability of pure, sufficient and well-characterized intrastrand cross-link lesion-bearing ODN substrates for future biochemical and biophysical characterizations. This was also the first demonstration that the coupling of radical I with its 5′ neighboring guanine can occur in the presence of molecular oxygen, suggesting that the formation of this and other types of intrastrand cross-link lesions might have important implications in the cytotoxic effects of ROS.     

Processing of plant mitochondrial tRNAGly and tRNASer(GCU) is independent of RNA editing

The genes encoding pea and potato mitochondrial tRNA^Gly and pea mitochondrial tRNA^Ser(GCU) were analyzed with particular respect to their expression. Secondary-structure models deduced from the identical potato and pea tRNA^Gly gene sequences revealed A₇:C₆₆ mismatches in the seventh base pair at the base of the acceptor stems of both tRNAs. Sequence analyses of tRNA^Gly cDNA clones showed that these mispairings are not corrected by C₆₆ to U₆₆ conversions, as observed in plant mitochondrial tRNA^Phe. Likewise, a U₆:C₆₇ mismatch identified in the acceptor stem of the pea tRNA^Ser(GCU) is not altered by RNA editing to a mismatched U:U pair, which is created by RNA editing in Oenothera mitochondrial tRNA^Cys. In vitro processing reactions with the respective tRNA^Gly and tRNA^Ser(GCU) precursors show that such conversions are not necessary for 5′ and 3′ end maturation of these tRNAs. These results demonstrate that not all C:A (A:C) or U:C (C:U) mismatches in double-stranded regions of tRNAs are altered by RNA editing. An RNA editing event in plant mitochondrial tRNAs is thus not generally indicated by the presence of a mismatch but may depend on additional parameters.     

Oligoribonucleotides containing 2',5'-phosphodiester linkages exhibit binding selectivity for 3',5'-RNA over 3',5'-ssDNA.

Oligoribonucleotides containing 2',5'-phosphodiester linkages have been synthesized on a solid support by the 'silyl-phosphoramidite' method. The stability of complexes formed between these oligonucleotides and complementary 3',5'-RNA strands have been studied using oligoadenylates and a variety of oligonucleotides of mixed base sequences including phosphorothioate backbones. In many cases, particularly for 2',5'-linked adenylates, the UV melting profiles are quite sharp and exhibit large hyperchromic changes. Substituting a few 3',5'-linkages with the 2',5'-linkage within an oligomer lowers the Tm of the complex and the degree of destabilization depends on the neighboring residues and neighboring linkages. The 2',5'-linked oligoribonucleotides prepared in this study exhibited remarkable selectivity for complementary single stranded RNA over DNA. For example, in 0.01 M phosphate buffer--0.10 M NaCl (pH 7.0), no association was observed between 2',5'-r(CCC UCU CCC UUC U) and its Watson-Crick DNA complement 3',5'-d(AGAAGGGAGAGGG). However, 2',5'-r(CCC UCU CCC UUC U) with its RNA complement 3',5'-r(AGAAGGGAGAGGG) forms a duplex which melts at 40 degrees C. The decamer 2',5'-r(Ap)9A forms a complex with both poly dT and poly rU but the complex [2',5'-r(Ap)9A]:[poly dT] is unstable (Tm, -1 degree C) and is seen only at high salt concentrations. In view of their unnatural character and remarkable selectivity for single stranded RNA, 2',5'-oligo-RNAs and their derivatives may find use as selective inhibitors of viral mRNA translation, and as affinity ligands for the purification of cellular RNA.     

The nucleotide sequences of tRNASer (GCU) and tRNAGln (UUG) genes from tobacco chloroplasts.

The nucleotide sequence of tobacco chloroplast genes for tRNASer (GCU) and tRNAGln (UUG) have been determined. These tRNA genes are encoded on the same DNA strand and separated by 1144 bp. Two open reading frames of 52 codons and 98 codons have been found in this spacer region. The tRNASer (GCU) and tRNAGln (UUG) deduced from the DNA sequences show 67% and 76% sequence homologies with E. coli tRNASer (GCU) and tRNAGln (UUG), respectively.     

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.     

Energetic and conformational contributions to the stability of Okazaki fragments

A combination of spectroscopic and calorimetric techniques was used to determine complete thermodynamic profiles accompanying the folding of a model Okazaki fragment with sequence 5'-r(gagga)d(ATCTTTG)-3'/5'-d(CAAAGATTCCTC)-3' and control DNA (with and without thymidine substitutions for uridine), RNA, and hybrid duplexes. Circular dichroism spectroscopy indicated that all DNA duplexes are in the B conformation, the RNA and hybrid duplexes are in the A conformation, and the Okazaki fragment exhibits a spectrum between the A and B conformations. Ultraviolet and differential scanning calorimetry melting experiments reveal that all duplexes unfold in two-state transitions with thermal stabilities that follow the order RNA > OKA > DNA (with thymidines) > hybrids > DNA (with uridines). The dependence of the transition temperature on salt concentration yielded counterion releases in the following order: DNA (with thymidines) > RNA > DNA (with uridines) > OKA > hybrids. Thus, Okazaki fragments have a conformation and charge density between those of its components DNA and hybrid segments. However, the presence of the RNA-DNA/DNA junction confers on them higher stabilities than their component hybrid and DNA segments. The binding of intercalators to an Okazaki hairpin of sequence 5'-r(gc)d(GCU5GCGC)-3' and to its control DNA hairpin has also been studied. The results show that the binding of intercalators to Okazaki fragments is accompanied with higher heats and lower binding affinities, compared with DNA duplexes. This suggests that the presence of an RNA/DNA junction yields a larger surface contact to interact with the phenanthroline ring of the intercalators, which may lead to a larger disruption of the flexible flanking bases of the junction. The overall results suggest that the presence of this junction stabilizes Okazaki fragments and provides a structural feature that can be exploited in the design of drugs to specifically target these molecules.     

Mechanisms of DNA damage by chromium(V) carcinogens.

Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3'-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.     

An EPR study of the transfer and trapping of holes produced by radiation in guanine(thioguanine) hydrochloride single crystals

Single crystals of guanine hydrochloride monohydrate, guanine hydrochloride dihydrate and anhydrous guanine dihydrochloride, doped with thioguanine, were irradiated with X and gamma rays. In all three systems the dominant radicals were associated with thioguanine. In the former two systems the stabilized species is the thiyl radical, formed by initial loss of an electron at some of the guanines in the crystal lattice, followed by hole migration to thioguanine and subsequent deprotonation of the radical formed. In the anhydrous guanine(thioguanine) dihydrochloride, that process is followed by acquisition of a chlorine ion. In the guanine hydrochloride monohydrate and guanine hydrochloride dihydrate lattices, systems of interacting closely spaced stacked bases and strings of chloride ions might support the migration of electrons and/or holes. In anhydrous guanine dihydrochloride, neither the bases nor the Cl- ions alone are capable of providing the means for the long-range electron, energy and spin transfer. It is the interchangeable sequence of the charged bases and the Cl- ions that makes the supporting strings or networks. The ultimate chlorination of the thioguanine-centered electron-loss radicals depends mainly on the availability of the Cl- ions and the space for their accommodation in the vicinity of the sulfur atom.     

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.     

Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO₃^·− radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO₃^·− radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpC_npT) and 5′-d(TpC_npG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO₃^·− radicals and enzymatic excision of the 5′-d(G^*pT^*) product.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.     

One-electron oxidation of beta-amyloid peptide: sequence modulation of reactivity

Amyloid beta peptide (Abeta) is a 39 to 43 amino-acid-long peptide implicated in Alzheimer's disease. One of its mechanisms of toxicity is related to its redox properties. Therefore we studied its one electron oxidation using azide free radicals produced in gamma and pulse radiolysis, and compared the results with those obtained with the reverse sequence Abeta(40-1). HPLC analysis combined with absorption, fluorescence, Raman spectroscopy, and MALDI-TOF MS were used for product identification. Met35 was shown to be the target in Abeta(1-40); oxidation leads to a major compound that is Abeta with methionine sulfoxide. Similarly, oxidation of fragment Abeta(29-40) also leads to methionine sulfoxide. For Abeta(40-1), Met35 is not reactive and Tyr10 is the target of azide radicals. The major products are peptide dimer linked by dityrosine and trimer. The lowering of the one-electron reduction potential of the MetS+/Met couple, which was proposed, is in agreement with our findings. To our knowledge, this is the first time that such a drastic effect of the primary sequence is observed in a small peptide. In addition, it is also the first experimental demonstration of the sensitivity of the one-electron reduction potential of methionine on neighboring groups.     

Radiation-activated nuclease activity of o,o'-Diphenyleneiodonium cations (DPI): a reductively initiated chain reaction involving the C1' chemistry

o,o'-Diphenyleneiodonium cations (DPI) convert relatively harmless radiation-produced electrons into efficient DNA cleaving agents. The cleavage products are unaltered DNA bases, 5-methylenefuranone (5-MF), and a complete set of 3' and 5'-phosphorylated DNA fragments. The production of alkali-labile sites is a minor factor in the process. Based on the production of 5-MF, it is concluded that DNA cleavage by DPI cations involves (but may not be limited to) the C1' chemistry. The loss of 3-aminoDPI (ADPI) cations bound to highly polymerized calf thymus DNA appears to be due to a short-chain reaction with an apparent length of up to 2.1 ADPI cations consumed for each radiation-produced electron. The suggested chain reaction mechanism includes the one-electron oxidation of DNA radicals (including the C1' sugar radical) by ADPI cations bound to the same duplex. The yields of DNA loss in complexes formed by ADPI with short synthetic duplexes indicate that there is more than a 60% probability of DNA damage after one-electron reduction of ADPI.     

Identification by electron spin resonance spectroscopy of free radicals produced during autoxidative melanogenesis

Free radicals produced during the autoxidation of 3,4-dihydroxyphenylalanine (DOPA) and other catechol(amine)s to melanins have been studied using electron spin resonance spectroscopy. Magnetic parameters for the radical intermediates have been determined, allowing the radicals to be unambiguously identified. Three types of radical are formed: the primary radical from one-electron oxidation of the parent catechol(amine); and two secondary radicals, one formed via OH- substitution, the other via cyclization. The formation of these radical species can be linked to molecular products formed during catecholamine oxidation and melanin formation.     

LC-MS/MS identification and yeast polymerase eta bypass of a novel gamma-irradiation-induced intrastrand cross-link lesion G[8-5]C

Reactive oxygen species can give rise to intrastrand cross-link lesions, where two neighboring nucleobases are covalently bonded. Here, we employed LC-MS/MS and demonstrated for the first time that gamma irradiation of a synthetic duplex oligodeoxyribonucleotide can give rise to an intrastrand cross-link lesion G[8-5]C, where the C8 carbon atom of guanine and the C5 carbon atom of its 3'-neighboring cytosine are covalently bonded. We also carried out in vitro replication studies of a substrate containing a site-specifically incorporated G[8-5]C, and our results showed that yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion. Steady-state kinetic analyses for nucleotide incorporation by pol eta showed that the 3'-cytosine moiety of the cross-link did not significantly affect either the efficiency or the fidelity of nucleotide incorporation. The 5' guanine portion of the cross-link lesion, however, markedly reduced both the efficiency and the fidelity of nucleotide incorporation; the insertion of dGMP or dAMP was slightly favored over the insertion of the correct nucleotide, dCMP, which was in turn favored over the insertion of dTMP. The above results support that the oxidative cross-link lesion, if not repaired, can be mutagenic.     

Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine

The replacement of thymidine with 5-bromo-2′-deoxyuridine (^BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a ^BrdU or its closely related 5-bromo-2′-deoxycytidine (^BrdC). Our results showed that two types of crosslink products could be induced from d(^BrCG), d(^BrUG), d(G^BrU), or d(A^BrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5′ flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5′ neighboring nucleoside was a dG than while it was a dA, and ^BrdC induced the formation of crosslink products much more efficiently than ^BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.