Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-Cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'-deoxyguanosine

Mutations in breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 predispose women to a high risk of these cancers. Here, we show that lymphoblasts of women with BRCA1 mutations who had been diagnosed with breast cancer are deficient in the repair of some products of oxidative DNA damage, namely, 8-hydroxy-2'-deoxyguanosine and 8,5'-cyclopurine-2'-deoxynucleosides. Cultured lymphoblasts from 10 individuals with BRCA1 mutations and those from 5 control individuals were exposed to 5 Gy of ionizing radiation to induce oxidative DNA damage and then allowed to repair this damage. DNA samples isolated from these cells were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure 8-hydroxy-2'-deoxyguanosine, (5'-S)-8,5'-cyclo-2'-deoxyadenosine, (5'-R)-8,5'-cyclo-2'-deoxyguanosine, and (5'-S)-8,5'-cyclo-2'-deoxyguanosine. After irradiation and a subsequent period of repair, no significant accumulation of these lesions was observed in the DNA from control cells. In contrast, cells with BRCA1 mutations accumulated statistically significant levels of these lesions in their DNA, providing evidence of a deficiency in DNA repair. In addition, a commonly used breast tumor cell line exhibited the same effect when compared to a relevant control cell line. The data suggest that BRCA1 plays a role in cellular repair of oxidatively induced DNA lesions. The failure of cells with BRCA1 mutations to repair 8,5'-cyclopurine-2'-deoxynucleosides indicates the involvement of BRCA1 in nucleotide-excision repair of oxidative DNA damage. This work suggest that accumulation of these lesions may lead to a high rate of mutations and to deleterious changes in gene expression, increasing breast cancer risk and contributing to breast carcinogenesis.     

Identification and quantification of 8,5'-cyclo-2'-deoxy-adenosine in DNA by liquid chromatography/ mass spectrometry

Recent studies suggested that 8,5'-cyclo-2'-deoxyadenosine may play a role in diseases with defective nucleotide-excision repair. This compound is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the sugar moiety of 2'-deoxyadenosine. It is likely to be repaired by nucleotide-excision repair rather than by base-excision repair because of a covalent bond between the sugar and base moieties. We studied the measurement of 8,5'-cyclo-2'-deoxyadenosine in DNA by liquid chromatography/isotope-dilution mass spectrometry. A methodology was developed for the analysis of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography in DNA hydrolyzed to nucleosides by a combination of four enzymes, i.e., DNase I, phosphodiesterases I and II, and alkaline phosphatase. Detection by mass spectrometry was performed using atmospheric pressure ionization-electrospray process in the positive ionization mode. Results showed that liquid chromatography/isotope-dilution mass spectrometry is well suited for identification and quantification of 8,5'-cyclo-2'-deoxyadenosine in DNA. Both (5'R)- and (5'S)-diastereomers of 8,5'-cyclo-2'-deoxyadenosine were detected. The level of sensitivity of liquid chromatography/mass spectrometry with selected-ion monitoring amounted to 2 fmol of this compound on the column. The yield of 8,5'-cyclo-2'-deoxyadenosine was measured in DNA in aqueous solution exposed to ionizing radiation at doses from 2.5 to 80 Gray. Gas chromatography/mass spectrometry was also used to measure this compound in DNA. Both techniques yielded similar results. The yield of 8,5'-cyclo-2'-deoxyadenosine was comparable to the yields of some of the other major modified bases in DNA, which were measured using gas chromatography/mass spectrometry. The measurement of 8,5'-cyclo-2'-deoxyadenosine by liquid chromatography/mass spectrometry may contribute to the understanding of its biological properties and its role in diseases with defective nucleotide-excision repair.     

Mass spectrometric assays for the tandem lesion 8,5'-cyclo-2'-deoxyguanosine in mammalian DNA

8,5'-Cyclopurine 2'-deoxynucleosides are among the major lesions in DNA that are formed by attack of hydroxyl radical. These compounds represent a concomitant damage to both sugar and base moieties of the same nucleoside and thus can be considered tandem lesions. Because of the presence of a covalent bond between the sugar and purine moieties, these tandem lesions are not repaired by base excision repair but by nucleotide excision repair. Thus, they may play a role in diseases with defective nucleotide excision repair. We recently reported the identification and quantification of 8,5'-cyclo-2'-deoxyadenosine (8,5'-cdAdo) in DNA by liquid chromatography/mass spectrometry with the isotope dilution technique (LC/IDMS) [Dizdaroglu, M., Jaruga, P., and Rodriguez, H. (2001) Free Radical Biol. Med. 30, 774-784]. In the present work, we investigated the measurement of 8,5'-cyclo-2'-deoxyguanosine (8,5'-cdGuo) in DNA by LC/IDMS. A methodology was developed for the separation of both (5'R)- and (5'S)-diastereomers of this compound in enzymic hydrolysates of DNA. The mass spectra were recorded using an atmospheric pressure ionization-electrospray process in the positive ionization mode. For quantification, stable isotope-labeled analogues of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were prepared and isolated by semipreparative LC to be used as internal standards. The sensitivity level of LC/MS in the selected ion monitoring mode (LC/MS-SIM) was determined to be approximately 15 fmol of these compounds on the LC column. The yield of 8,5'-cdGuo was measured in DNA exposed in aqueous solution to ionizing radiation at doses from 2.5 to 40 Gy. For comparison, gas chromatography/mass spectrometry with the isotope dilution technique (GC/IDMS) was also employed to measure both (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo in DNA. Both techniques yielded nearly identical results. The radiation chemical yield of 8,5'-cdGuo was similar to those of other major purine-derived lesions in DNA. The sensitivity level of GC/MS-SIM was determined to be significantly greater than that of LC/MS-SIM (1 vs 15 fmol). The background levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were measured in calf thymus DNA and in DNA samples isolated from three different types of cultured human cells. The levels of (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo were approximately 2 lesions/10(6) DNA nucleosides and 10 lesions/10(6) DNA nucleosides, respectively. No significant differences between tissues were observed in terms of these background levels. The results showed that both LC/IDMS and GC/IDMS are well suited for the sensitive detection and precise quantification of both (5'R)-8,5'-cdGuo and (5'S)-8,5'-cdGuo in DNA.     

Free-radical-induced formation of an 8,5''-cyclo-2''-deoxyguanosine moiety in deoxyribonucleic acid.

Isolation and identification of a novel .OH-induced product, namely an 8,5'-cyclo-2'-deoxyguanosine moiety, in DNA and 2'-deoxyguanosine are described. .OH radicals were generated in dilute aqueous solutions by gamma-irradiation. Analyses of 2'-deoxyguanosine and enzymic hydrolysates of DNA by gas chromatography-mass spectrometry (g.c.-m.s.) after trimethylsilylation showed the presence of 8,5-cyclo-2'-deoxyguanosine on the basis of its fragment ions. This product was isolated by h.p.l.c. Its u.v. and n.m.r. spectra taken were in agreement with the structure suggested by its mass spectrum. Exact masses of the typical ions from the mass spectrum of the trimethylsilyl derivative of this product were measured by high-resolution m.s. The values found were in excellent agreement with the theoretical mass derived from the suggested fragmentation patterns. Both (5'R)- and (5'S)-epimers of 8,5'-cyclo-2'-deoxyguanosine were observed. These two diastereomers were separated from each other by g.c. as well as by h.p.l.c. The assignment of the epimers was accomplished on the basis of the n.m.r. data. The formation of 8,5'-cyclo-2'-deoxyguanosine was suppressed by the presence of O2 in the solutions. The use of g.c.-m.s. with the selected-ion monitoring technique facilitated the detection of 8,5'-cyclo-2'-deoxyguanosine in DNA at radiation doses as low as 1 Gy. Its mechanism of formation probably involves hydrogen atom abstraction by .OH radicals from the C-5' of the 2'-deoxyguanosine moiety followed by intramolecular cyclization with the formation of a covalent bond between the C-5' and C-8 and subsequent oxidation of the resulting N-7-centred radical.     

A 32P-postlabeling assay for the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine in mammalian tissues: evidence that four type II I-compounds are dinucleotides containing the lesion in the 3' nucleotide.

8,5'-Cyclopurine-2'-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of (32)P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5'-cyclo-2'-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic (32)P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The (32)P-postlabeling technique used here is capable of detecting 1-5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.     

DNA damage products (5'R)- and (5'S)-8,5'-cyclo-2'-deoxyadenosines as potential biomarkers in human urine for atherosclerosis

We hypothesized that DNA damage products (5'R)-8,5'-cyclo-2'-deoxyadenosine (R-cdA) and (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA) may be well-suited biomarkers of risk and diagnosis for atherosclerosis. We tested this hypothesis by measuring the levels of R-cdA and S-cdA and another product, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), in urine of atherosclerosis patients and healthy individuals using liquid chromatography-tandem mass spectrometry with isotope dilution. We showed the presence of these products at significantly greater concentrations in urine of atherosclerosis patients than in that of healthy individuals. Our data suggest that R-cdA and S-cdA can be accurately and reproducibly measured in human urine as potential biomarkers of risk and diagnosis for atherosclerosis.     

(5'S)-8,5'-cyclo-2'-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli

8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.     

Mutation spectra induced by replication of two vicinal oxidative DNA lesions in mammalian cells

Ionizing radiations often induce multiple and clustered DNA lesions at the site of DNA interaction. As a model, we have studied the toxicity and the mutagenicity of two adjacent oxidative bases as clustered DNA lesions in mammalian cells using shuttle vectors. The chosen oxidative lesions were 8-oxo-7,8-dihydroguanine, the formylamine residue resulting from the oxidation of a pyrimidine base and the tandem lesion 8-oxo-7,8-dihydroguanine/formylamine where both modifications are located at a vicinal position. A single-stranded DNA shuttle vector carrying a unique DNA lesion was constructed, transfected into simian COS7 cells and mutations induced after replication in mammalian cells were screened in bacteria. 8-oxo-7,8-dihydroguanine, as expected, does not affect greatly survival (70% bypass) whereas formylamine and the tandem lesions are blocking alterations, DNA polymerase bypass being of 45% and 17%, respectively. Base insertion opposite the lesion was studied. Under our experimental conditions, replication of 8-oxo-7, 8-dihydroguanine finally gives rise to guanine:cytosine pairing, rendering this lesion only slightly mutagenic. This is not the case for the formylamine that codes preferentially for adenine (71%). In addition, one-base deletions were observed targeted to the site to the lesion. Cytosine and thymine were inserted opposite the lesion with similar but low frequencies. Thus, coding properties of the formylamine render this residue very mutagenic when coming from the oxidative alteration of a cytosine. The coding properties of the tandem damage are a combination of the contribution of the two isolated lesions with a very high percentage of adenine insertion (94%) opposite the formylamine residue of the tandem lesion. The toxicity as well as the mutation spectrum of the tandem lesion allow us to speculate about the molecular mechanism with which the DNA polymerase replicates these two lesions.     

Fidelity of replication of the leading and the lagging DNA strands opposite N-methyl-N-nitrosourea-induced DNA damage in human cells.

Semi-conservative replication of double-stranded DNA in eukaryotic cells is an asymmetric process involving leading and lagging strand synthesis and different DNA polymerases. We report a study to analyze the effect of these asymmetries when the replication machinery encounters alkylation-induced DNA adducts. The model system is an EBV-derived shuttle vector which replicates in synchrony with the host human cells and carries as marker gene the bacterial gpt gene. A preferential distribution of N-methyl-N-nitrosourea (MNU)-induced mutations in the non transcribed DNA strand of the shuttle vector pF1-EBV was previously reported. The hypermutated strand was the leading strand. To test whether the different fidelity of DNA polymerases synthesizing the leading and the lagging strands might contribute to MNU-induced mutation distribution the mutagenesis study was repeated on the shuttle vector pTF-EBV which contains the gpt gene in the inverted orientation. We show that the base substitution error rates on an alkylated substrate are similar for the replication of the leading and lagging strands. Moreover, we present evidence that the fidelity of replication opposite O6-methylguanine adducts of both the leading and lagging strands is not affected by the 3' flanking base. The preferential targeting of mutations after replication of alkylated DNA is mainly driven by the base at the 5' side of the G residues.     

Evidence for upregulated repair of oxidatively induced DNA damage in human colorectal cancer

Carcinogenesis may involve overproduction of oxygen-derived species including free radicals, which are capable of damaging DNA and other biomolecules in vivo. Increased DNA damage contributes to genetic instability and promote the development of malignancy. We hypothesized that the repair of oxidatively induced DNA base damage may be modulated in colorectal malignant tumors, resulting in lower levels of DNA base lesions than in surrounding pathologically normal tissues. To test this hypothesis, we investigated oxidatively induced DNA damage in cancerous tissues and their surrounding normal tissues of patients with colorectal cancer. The levels of oxidatively induced DNA lesions such as 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine and (5'S)-8,5'-cyclo-2'-deoxyadenosine were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution tandem mass spectrometry. We found that the levels of these DNA lesions were significantly lower in cancerous colorectal tissues than those in surrounding non-cancerous tissues. In addition, the level of DNA lesions varied between colon and rectum tissues, being lower in the former than in the latter. The results strongly suggest upregulation of DNA repair in malignant colorectal tumors that may contribute to the resistance to therapeutic agents affecting the disease outcome and patient survival. The type of DNA base lesions identified in this work suggests the upregulation of both base excision and nucleotide excision pathways. Development of DNA repair inhibitors targeting both repair pathways may be considered for selective killing of malignant tumors in colorectal cancer.     

Comparative Evaluation of DNA Extraction Methods from Feces of Multiple Host Species for Downstream Next-Generation Sequencing

The gastrointestinal tract contains a vast community of microbes that to this day remain largely unculturable, making studies in this area challenging. With the newly affordable advanced sequencing technology, important breakthroughs in this exciting field are now possible. However, standardized methods of sample collection, handling, and DNA extraction have yet to be determined. To help address this, we investigated the use of 5 common DNA extraction methods on fecal samples from 5 different species. Our data show that the method of DNA extraction impacts DNA concentration and purity, successful NGS amplification, and influences microbial communities seen in NGS output dependent on the species of fecal sample and the DNA extraction method used. These data highlight the importance of careful consideration of DNA extraction method used when designing and interpreting data from cross species studies.     

Time and concentration dependence of Fenton-induced oxidation of dG

The influence of incubation time and Fenton reagent concentrations was investigated on the oxidation of 2'-deoxyguanosine. The compounds identified and quantified, through use of an LC-MS/MS system, were 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8,5'-cyclo-2'-deoxyguanosine (8,5'cyclodG) and the secondary oxidation products guanidinohydantoin and dehydro-guanidinohydantoin. 8-oxodG and 8,5' cyclodG formed very quickly, reaching a maximum rapidly, but with 8-oxodG a rapid decline occurred thereafter due to its further oxidation into the secondary products, which formed more slowly. Due to the better stability, 8,5' cyclodG correlated better with the general level of oxidation than 8-oxodG. The results emphasize the advantages of measuring other oxidation adducts than 8-oxodG alone.     

Beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing

The diverse sequences of viral populations within individual hosts are the starting material for selection and subsequent evolution of RNA viruses such as foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the inoculum used to initiate experimental infection. Genomic sequences were determined in duplicate sequencing runs, and the consensus sequence of the inoculum determined by NGS was identical to that previously determined using the Sanger method. However, NGS revealed the fine polymorphic substructure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. Some of the higher-frequency polymorphisms identified encoded changes within codons associated with heparan sulfate binding and were present in both foot lesions, revealing intermediate stages in the evolution of a tissue culture-adapted virus replicating within a mammalian host. We identified 2,622, 1,434, and 1,703 polymorphisms in the inoculum and in the two foot lesions, respectively: most of the substitutions occurred in only a small fraction of the population and represented the progeny from recent cellular replication prior to onset of any selective pressures. We estimated the upper limit for the genome-wide mutation rate of the virus within a cell to be 7.8 × 10(-4) per nucleotide. The greater depth of detection achieved by NGS demonstrates that this method is a powerful and valuable tool for the dissection of FMDV populations within hosts.     

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells.

We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagenicity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T----C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T----A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than that expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.