8-5--cyclo-2--deoxyadenosine and 8-5--cyclo-2--deoxyguanosine

Patients with the genetic disease xeroderma pigmentosum (XP) who lack the capacity to carry out nucleotides excision repair (NER) have a dramatically elevated risk of skin cancer on sun exposed areas of the body. NER is the DNA repair mechanism responsible for the removal of DNA lesions resulting from ultraviolet light. In addition, a subset of XP patients develop a progressive neurodegenerative disease, referred to as XP neurologic disease, which is thought to be the result of accumulation of endogenous DNA lesions that are repaired by NER but not other repair pathways. The 8,5-cyclopurine deoxynucleotides (cyPu) have emerged as leading candidates for such lesions, in that they result from the reaction of the hydroxyl radical with DNA, are strong blocks to transcription in human cells, and are repaired by NER but not base excision repair. Here I present a focused perspective on progress into understating the repair and biological effects of these lesions. In doing so, I emphasize the role of Tomas Lindahl and his laboratory in stimulating cyPu research. I also include a critical evaluation of the evidence supporting a role for cyPu lesions in XP neurologic disease, with a focus on outstanding questions, and conceptual and technologic challenges.

Topics: Animals; Deoxyadenosines; Deoxyguanosine; DNA; DNA Damage; DNA Repair; Humans; Hydroxyl Radical; Neurodegenerative Diseases; Risk; RNA Polymerase II; Skin Neoplasms; Transcription, Genetic; Ultraviolet Rays; Xeroderma Pigmentosum

5',8-Cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine in their 5'R and 5'S diastereomeric forms are tandem-type lesions observed among the DNA modifications and identified in mammalian cellular DNA in vivo. These lesions result from the chemistry of the C5' radicals generated by the attack of HO˙ radicals to 2-deoxyribose units. Quantitative determination of these lesions in biological samples as biomarkers of free radical damage is a challenge. Results reported for irradiated samples of calf thymus DNA have been critically reviewed, underlining the need of further research for the potential involvement of these lesions in human health (76 references).

Topics: Cyclization; Deoxyadenosines; Deoxyguanosine; DNA Damage; Exoribonucleases; Hydroxyl Radical; Oxidation-Reduction; Stereoisomerism

8,5'-Cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) are among the major lesions formed in DNA by hydroxyl radical attack on 2'-deoxyadenosine and 2'-deoxyguanosine, respectively, followed by intramolecular cyclization between C5' and C8. Mechanisms of formation of these unique tandem lesions were elucidated. The 8,5'-cyclization causes an unusual puckering of the sugar moiety giving rise to significant distortion in the DNA double helix. Methodologies were developed for the measurement of these lesions in DNA by mass spectrometry coupled either with gas chromatography or high performance liquid chromatography. Both techniques allowed identification and quantification of both R- and S-diastereomers of cdA and cdG in DNA in vitro and in vivo. Because of the 8,5'-covalent bond between the sugar and base moieties in the same nucleoside, cdA and cdG are repaired by nucleotide excision repair rather than by base excision repair. Thus, these lesions may play a role in diseases with defective nucleotide excision repair. Their biological effects include blocking DNA polymerases, inhibition of gene expression, transcriptional mutagenesis among others. Accumulation of cdA and cdG was observed in tissues in vivo in connection to disease and environmental conditions, suggesting an important role for these lesions in disease processes including carcinogenesis and neuronal death.

Topics: Animals; Chromatography, Liquid; Deoxyadenosines; Deoxyguanosine; DNA; DNA Repair; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation; Humans; Hydroxyl Radical; Mass Spectrometry; Mice; Nucleic Acid Conformation

Topics: Animals; Deoxyadenosines; Deoxyguanosine; DNA; Gamma Rays; HeLa Cells; Humans; Liver; MCF-7 Cells; Mice; Oxidation-Reduction; Rats; Stereoisomerism

Reactive oxygen species generate many lesions in DNA, including R and S diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG). Herein, the result of replication of a plasmid containing S-cdA in Escherichia coli is reported. S-cdA was found mutagenic and highly genotoxic. Viability and mutagenicity of the S-cdA construct were dependent on functional pol V, but mutational frequencies (MFs) and types varied in pol II- and pol IV-deficient strains relative to the wild-type strain. Both S-cdA → T and S-cdA → G substitutions occurred in equal frequency in wild-type E. coli, but the frequency of S-cdA → G dropped in pol IV-deficient strain, especially when being SOS induced. This suggests that pol IV plays a role in S-cdA → G mutations. MF increased significantly in pol II-deficient strain, suggesting pol II's likely role in error-free translesion synthesis. Primer extension and steady-state kinetic studies using pol IV, exo-free Klenow fragment (KF (exo(-))), and Dpo4 were performed to further assess the replication efficiency and fidelity of S-cdA and S-cdG. Primer extension by pol IV mostly stopped before the lesion, although a small fraction was extended opposite the lesion. Kinetic studies showed that pol IV incorporated dCMP almost as efficiently as dTMP opposite S-cdA, whereas it incorporated the correct nucleotide dCMP opposite S-cdG 10-fold more efficiently than any other dNMP. Further extension of each lesion containing pair, however, was very inefficient. These results are consistent with the role of pol IV in S-cdA → G mutations in E. coli. KF (exo(-)) was also strongly blocked by both lesions, but it could slowly incorporate the correct nucleotide opposite them. In contrast, Dpo4 could extend a small fraction of the primer to a full-length product on both S-cdG and S-cdA templates. Dpo4 incorporated dTMP preferentially opposite S-cdA over the other dNMPs, but the discrimination was only 2- to 8-fold more proficient. Further extension of the S-cdA:T and S-cdA:C pair was not much different. For S-cdG, conversely, the wrong nucleotide, dTMP, was incorporated more efficiently than dCMP, although one-base extension of the S-cdG:T pair was less efficient than the S-cdG:C pair. S-cdG, therefore, has the propensity to cause G → A transition, as was reported to occur in E. coli. The results of this study are consistent with the strong replication blocking nature of S-cdA and S-cdG, and their ability to initiate error-pron

Topics: Deoxyadenosines; Deoxyguanosine; DNA Polymerase beta; DNA Polymerase I; Escherichia coli; Escherichia coli Proteins; Mutagens; Nucleotides

The hydroxyl radical is a powerful oxidant that generates DNA lesions including the stereoisomeric R and S 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) pairs that have been detected in cellular DNA. Unlike some other oxidatively generated DNA lesions, cdG and cdA are repaired by the human nucleotide excision repair (NER) apparatus. The relative NER efficiencies of all four cyclopurines were measured and compared in identical human HeLa cell extracts for the first time under identical conditions, using identical sequence contexts. The cdA and cdG lesions were excised with similar efficiencies, but the efficiencies for both 5'R cyclopurines were greater by a factor of ∼2 than for the 5'S lesions. Molecular modeling and dynamics simulations have revealed structural and energetic origins of this difference in NER-incision efficiencies. These lesions cause greater DNA backbone distortions and dynamics relative to unmodified DNA in 5'R than in 5'S stereoisomers, producing greater impairment in van der Waals stacking interaction energies in the 5'R cases. The locally impaired stacking interaction energies correlate with relative NER incision efficiencies, and explain these results on a structural basis in terms of differences in dynamic perturbations of the DNA backbone imposed by the R and S covalent 5',8 bonds.

Topics: Deoxyadenosines; Deoxyguanosine; DNA; DNA Damage; DNA Repair; HeLa Cells; Humans; Molecular Dynamics Simulation; Nucleic Acid Conformation; Stereoisomerism

8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases--Escherichia coli Xth and human APE1--can remove 5'S cdA (S-cdA) at 3' termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3' end. To understand the structural basis of 3' repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2'-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3'-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3'→5' exonuclease function and its inhibition by 3'-terminal S-cdA residue. This structure was used as a template to model 3'-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3' repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3' DNA termini in E. coli and human cells, respectively.

Topics: Denaturing Gradient Gel Electrophoresis; Deoxyadenosines; Deoxyguanosine; Deoxyribonuclease IV (Phage T4-Induced); DNA Adducts; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Escherichia coli; Escherichia coli Proteins; Exonucleases; Humans; Models, Molecular; Molecular Structure; Oligonucleotides; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; X-Ray Diffraction; Yeasts

Exposure of aqueous solutions of DNA to X- or γ-rays, which induces the hydroxyl radical as one of the major reactive oxygen species (ROS), can result in the generation of a battery of single-nucleobase and bulky DNA lesions. These include the (5'R) and (5'S) diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG), which were also found to be present at appreciable levels in DNA isolated from mammalian cells and tissues. However, it remains unexplored how efficiently the cdA and cdG can be induced by Fenton-type reagents. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) with the use of the isotope-dilution technique, here we demonstrated that treatment of calf thymus DNA with Cu(II) or Fe(II), together with H2O2 and ascorbate, could lead to dose-responsive formation of both the (5'R) and (5'S) diastereomers of cdA and cdG, though the yields of cdG were 2-4 orders of magnitude lower than that of 8-oxo-7,8-dihydro-2'-deoxyguanosine. This result suggests that the Fenton reaction may constitute an important endogenous source for the formation of the cdA and cdG. Additionally, the (5'R) diastereomers of cdA and cdG were induced at markedly higher levels than the (5'S) counterparts. This latter finding, in conjunction with the previous observations of similar or greater levels of the (5'S) than (5'R) diastereomers of the two lesions in mammalian tissues, furnishes an additional line of evidence to support the more efficient repair of the (5'R) diastereomers of the purine cyclonucleosides in mammalian cells.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Deoxyadenosines; Deoxyguanosine; DNA; Dose-Response Relationship, Drug; Hydrogen Peroxide; Iron; Structure-Activity Relationship; Tandem Mass Spectrometry

Reactive oxygen species can give rise to a battery of DNA damage products including the 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) tandem lesions. The 8,5'-cyclopurine-2'-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNA replication in mammalian cells. Previous in vitro biochemical assays have suggested a role for human polymerase (Pol) η in the insertion step of translesion synthesis (TLS) across the (5'S) diastereomers of cdA and cdG. Using in vitro steady-state kinetic assay, herein we showed that human Pol ι and a two-subunit yeast Pol ζ complex (REV3/REV7) could function efficiently in the insertion and extension steps, respectively, of TLS across S-cdA and S-cdG; human Pol κ and Pol η could also extend past these lesions, albeit much less efficiently. Results from a quantitative TLS assay showed that, in human cells, S-cdA and S-cdG inhibited strongly DNA replication and induced substantial frequencies of mutations at the lesion sites. Additionally, Pol η, Pol ι, and Pol ζ, but not Pol κ, had important roles in promoting replication through S-cdA and S-cdG in human cells. Based on these results, we propose a model for TLS across S-cdA and S-cdG in human cells, where Pol η and/or Pol ι carries out nucleotide insertion opposite the lesion, whereas Pol ζ executes the extension step.

Topics: Base Sequence; Deoxyadenosines; Deoxyguanosine; DNA; DNA Adducts; DNA Damage; DNA Polymerase iota; DNA Replication; DNA-Directed DNA Polymerase; Gene Knockdown Techniques; Humans; Kinetics; Molecular Sequence Data; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Saccharomyces cerevisiae

Thiopurines are among the most successful chemotherapeutic agents used for treating various human diseases, including acute lymphoblastic leukemia and chronic inflammation. Although metabolic conversion and the subsequent incorporation of 6-thioguanine ((S)G) nucleotides into nucleic acids are considered important for allowing the thiopurine drugs to induce their cytotoxic effects, alternative mechanisms may also exist. We hypothesized that an unbiased analysis of (S)G-induced perturbation of the entire proteome might uncover novel mechanism(s) of action of the drug. We performed a quantitative assessment of global protein expression in control and (S)G-treated Jurkat T cells by employing stable isotope labeling by amino acids in cell culture and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS quantification results uncovered substantially decreased expression of a large number of proteins in the mitochondrial respiratory chain complex, and Ingenuity Pathway Analysis of the significantly altered proteins showed that (S)G treatment induced mitochondrial dysfunction. This was accompanied by diminished uptake of MitoTracker Deep Red and the elevated formation of oxidatively induced DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine. Together, our results suggested that (S)G may exert its cytotoxic effect by inducing mitochondrial dysfunction and reactive oxygen species formation in acute lymphoblastic leukemia cells.

Topics: Amino Acid Sequence; Antimetabolites, Antineoplastic; Chromatography, Liquid; Deoxyadenosines; Deoxyguanosine; DNA Damage; Gene Expression; Humans; Isotope Labeling; Jurkat Cells; Mitochondria; Mitochondrial Membranes; Molecular Sequence Annotation; Molecular Sequence Data; Oxidation-Reduction; Proteome; Reactive Oxygen Species; Tandem Mass Spectrometry; Thioguanine

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5' nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.

Topics: Base Pairing; Deoxyadenosines; Deoxyguanosine; DNA; DNA Adducts; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Humans; Kinetics; Saccharomyces cerevisiae

5'-R and 5'-S diastereoisomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24-32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N(2)-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N(2)-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.

Topics: Base Pairing; Deoxyadenosines; Deoxyguanosine; Deoxyribonuclease (Pyrimidine Dimer); DNA; DNA Glycosylases; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Escherichia coli Proteins; HeLa Cells; Humans; Nucleic Acid Heteroduplexes

The purpose of our study was to develop suitable methods to quantify oxidative DNA lesions in the setting of transition metal-related diseases. Transition metal-driven Fenton reactions constitute an important endogenous source of reactive oxygen species (ROS). In genetic diseases with accumulation of transition metal ions, excessive ROS production causes pathophysiological changes, including DNA damage. Wilson's disease is an autosomal recessive disorder with copper toxicosis due to deficiency of ATP7B protein needed for excreting copper into bile. The Long-Evans Cinnamon (LEC) rat bears a deletion in Atp7b gene and serves as an excellent model for hepatic Wilson's disease. We used a sensitive capillary liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS/MS) method in conjunction with the stable isotope-dilution technique to quantify several types of oxidative DNA lesions in the liver and brain of LEC rats. These lesions included 5-formyl-2'-deoxyuridine, 5-hydroxymethyl-2'-deoxyuridine, and the 5'R and 5'S diastereomers of 8,5'-cyclo-2'-deoxyguanosine and 8,5'-cyclo-2'-deoxyadenosine. Moreover, the levels of these DNA lesions in the liver and brain increased with age and correlated with age-dependent regulation of the expression of DNA repair genes in LEC rats. These results provide significant new knowledge for better understanding the implications of oxidative DNA lesions in transition metal-induced diseases, such as Wilson's disease, as well as in aging and aging-related pathological conditions.

Topics: Animals; Brain; Chromatography, High Pressure Liquid; Deoxyadenosines; Deoxyguanosine; Deoxyuridine; DNA Damage; DNA Repair; Gene Expression Regulation; Isotopes; Liver; Oxidation-Reduction; Rats; Rats, Inbred LEC; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Stereoisomerism; Tandem Mass Spectrometry; Thymidine; Time Factors

Radiation-induced degradation of purine and pyrimidine nucleosides gave rise to carbon-bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which modification of both the base and 2-deoxyribose has occurred. A solid-phase synthetic method was designed for the incorporation of both 5'R and 5'S diastereoisomers of 5',8-cyclopurine 2'-deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical and biophysical features of such lesions. We report the preparation of the phosphoramidite synthons of (5'R)-5', 8-cyclo-2'-deoxyadenosine (2), (5'S)-5',8-cyclo-2'-deoxyguanosine (3), and (5'R)-5',8-cyclo-2'-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then inserted into several oligonucleotides by automated procedures. Analysis of modified DNA oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and endonucleases confirmed the base compositions and the integrity of free radical-induced tandem lesions 2-4 that were chemically inserted.

Topics: Alkaline Phosphatase; Chromatography, High Pressure Liquid; Deoxyadenosines; Deoxyguanosine; Hydrolysis; Indicators and Reagents; Oligonucleotides; Phosphoric Diester Hydrolases; Single-Strand Specific DNA and RNA Endonucleases; Spectrophotometry, Ultraviolet; Stereoisomerism

Reactions of hydroxyl radicals with DNA form a variety of base and sugar products and 8,5'-cyclopurine 2'-deoxyribonucleoside residues in DNA. Here we report the effect of DNA conformation on the yields of 8,5'-cyclopurine 2'-deoxynucleosides and the ratios of their (5'R)- and (5'S)-diastereomers. Calf thymus DNA in native (double-stranded DNA) or heat-denatured form (single-stranded DNA) was exposed to hydroxyl radicals generated by ionizing radiation in nitrous oxide-saturated phosphate buffer. Doses ranging from 10 to 40 Gy were used to ensure low levels of damage to DNA and thus to preserve its secondary structure in experiments with double-stranded DNA (ds-DNA). After irradiation, DNA was hydrolysed enzymatically to 2'-deoxyribonucleosides. The hydrolysates were dried, trimethylsilylated, and analyzed by capillary gas chromatography-mass spectrometry with selected-ion monitoring. An internal standard was used for quantitative measurements and added to DNA samples prior to enzymatic hydrolysis. The yields of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in single-stranded DNA (ss-DNA) were higher than those in ds-DNA. The (5'R)-diastereomers of both compounds were found to predominate over their (5'S)-diastereomers in ss-DNA. In contrast, the yields of the (5'S)-diastereomers in ds-DNA were slightly higher than those of the (5'R)-diastereomers. The G values of 8,5'-cyclo-2'-deoxyadenosine in ss-DNA and ds-DNA were 0.042 and 0.025, respectively. Those of 8,5'-cyclo-2'-deoxyguanosine in ss-DNA and ds-DNA were 0.038 and 0.017, respectively.

Topics: Deoxyadenosines; Deoxyguanosine; Deoxyribonucleosides; DNA; Free Radicals; Hydroxides; Nucleic Acid Conformation