Site-specific DNA Damage Research Articles

Rosmarinic acid, a natural polyphenol, has a potential pro-oxidant risk via NADH-mediated oxidative DNA damage

BackgroundRosmarinic acid (RA) has a wide range of beneficial effects on human health. On the other hand, RA has been reported to induce metal-mediated reactive oxygen species (ROS) generation and DNA damage. However, its mechanism remains unknown. In this study, to clarify the underlying mechanism, we analyzed metal-mediated DNA damage in isolated DNA treated with RA and its analog isorinic acid.ResultsRA plus Cu(II), but not Fe(III), significantly increased 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) formation, an indicator of oxidative DNA damage, in calf thymus DNA. Furthermore, a comparison of the 8-oxodG formation induced by RA and its analog isorinic acid suggested that the catechol groups in RA could be associated with their abilities to form 8-oxodG. Interestingly, the 8-oxodG formation induced by RA and isorinic acid plus Cu(II) was markedly enhanced by the addition of NADH, an endogenous reductant. To elucidate the mechanism of RA plus Cu(II)-induced oxidative DNA damage, we examined DNA damage in 32P-labeled DNA treated with RA in the presence of Cu(II). RA plus Cu(II) caused DNA cleavage, which was enhanced by piperidine treatment, suggesting that RA causes not only DNA strand breakage but also base modification. RA plus Cu(II)-induced DNA damage was inhibited by catalase (H2O2 scavenger), bathocuproine (Cu(I) chelator), and methional (scavenger of a variety of ROS other than •OH) but not by typical •OH scavengers and SOD, indicating the involvement of H2O2, Cu(I), and ROS other than •OH. DNA cleavage site analysis showing RA-induced site-specific DNA damage (frequently at thymine and some cytosine residues) supports the involvement of ROS other than •OH, because •OH causes DNA cleavage without site specificity. Based on these results, Cu(I) and H2O2 generation with concomitant RA autoxidation could lead to the production of Cu(I)-hydroperoxide, which induces oxidative DNA damage. o-Quinone and o-semiquinone radicals are likely to be again reduced to RA by NADH, which dramatically increases oxidative DNA damage, particularly at low concentrations of RA.ConclusionsIn this study, physiologically relevant concentrations of RA effectively induced oxidative DNA damage in isolated DNA through redox cycle reactions with copper and NADH.

Site-specific genotoxicity of rubiadin: localization and histopathological changes in the kidneys of rats.

Rubiadin (Rub) is a genotoxic component of madder color (MC) that is extracted from the root of Rubia tinctorum L. MC induces renal tumors and preneoplastic lesions that are found in the proximal tubule of the outer stripe of the outer medulla (OSOM), suggesting that the renal carcinogenicity of MC is site specific. To clarify the involvement of Rub in renal carcinogenesis of MC, we examined the distribution of Rub in the kidney of male gpt delta rats that were treated with Rub for 28days. We used desorption electrospray ionization quadrupole time-of-flight mass spectrometry imaging (DESI-Q-TOF-MSI), along with the histopathological analysis, immunohistochemical staining, and reporter gene mutation assays of the kidney. DESI-Q-TOF-MSI revealed that Rub and its metabolites, lucidin and Rub-sulfation, were specifically distributed in the OSOM. Histopathologically, karyomegaly characterized by enlarged nuclear and microvesicular vacuolar degeneration occurred in proximal tubule epithelial cells in the OSOM. The ɤ-H2AX- and p21-positive cells were also found in the OSOM rather than the cortex. Although dose-dependent increases in gpt and Spi- mutant frequencies were observed in both the medulla and cortex, the mutant frequencies in the medulla were significantly higher. The mutation spectra of gpt mutants showed that A:T-T:A transversion was predominant in Rub-induced gene mutations, consistent with those of MC. Overall, the data showed that the distribution of Rub and its metabolites resulted in site-specific histopathological changes, DNA damage, and gene mutations, suggesting that the distribution of genotoxic components and metabolites is responsible for the site-specific renal carcinogenesis of MC.

Myricetin causes site-specific DNA damage via reactive oxygen species generation by redox interactions with copper ions.

Myricetin (MYR), found in tea and berries, may have preventive effects on diseases, including Alzheimer's disease and cancer. However, MYR is also a mutagen, inducing DNA damage in the presence of metal ions. We have studied the molecular mechanisms of DNA damage by MYR in the presence of Cu(II) (MYR+Cu). Using 32P-5'-end-labeled DNA fragments, we analyzed site-specific DNA damage caused by MYR+Cu. MYR+Cu caused concentration-dependent DNA strand breaks and base alterations, leading to cleavage of DNA at thymine, cytosine, and guanine nucleotides. Formation of the oxidative DNA damage indicator, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in calf thymus DNA was increased by MYR+Cu. The production of 8-oxodG in MYR-treated HL-60 cells was significantly higher than in HP100 cells, which are more resistant to H2O2 than are HL-60 cells. Reactive oxygen species (ROS) scavengers were used to elucidate the mechanism of DNA damage. DNA damage was not inhibited by typical free hydroxyl radical (•OH) scavengers such as ethanol, mannitol, or sodium formate. However, methional, catalase, and bathocuproine inhibited DNA damage induced by MYR+Cu. These results suggest that H2O2, Cu(I), and ROS other than •OH are involved in MYR+Cu-induced DNA damage. We conclude that the Cu(I)/Cu(II) redox cycle and concomitant H2O2 production via autoxidation of MYR generate a complex of H2O2 and Cu(I), probably Cu(I)-hydroperoxide, which induces oxidative DNA damage.

Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage.

Eukaryotic DNA exists in chromatin, where the genomic DNA is packaged into a fundamental repeating unit known as the nucleosome. In this chromatin environment, our genomic DNA is constantly under attack by exogenous and endogenous stressors that can lead to DNA damage. Importantly, this DNA damage must be repaired to prevent the accumulation of mutations and ensure normal cellular function. To date, most in-depth biochemical studies of DNA repair proteins have been performed in the context of free duplex DNA. However, chromatin can serve as a barrier that DNA repair enzymes must navigate in order find, access, and process DNA damage in the cell. To facilitate future studies of DNA repair in chromatin, we describe a protocol for generating nucleosome containing site-specific DNA damage that can be utilized for a variety of in vitro applications. This protocol describes several key steps including how to generate damaged DNA oligonucleotides, the expression and purification of recombinant histones, the refolding of histone complexes, and the reconstitution of nucleosomes containing site-specific DNA damage. These methods will enable researchers to generate nucleosomes containing site-specific DNA damage for extensive biochemical and structural studies of DNA repair in the nucleosome.

Technological Advancements in External Beam Radiation Therapy (EBRT): An Indispensable Tool for Cancer Treatment.

Recent technological advancements have increased the efficacy of radiotherapy, leading to effective management of cancer patients with enhanced patient survival and improved quality of life. Several important developments like multileaf collimator, integration of imaging techniques like positron emission tomography (PET) and computed tomography (CT), involvement of advanced dose calculation algorithms, and delivery techniques have increased tumor dose distribution and decreased normal tissue toxicity. Three-dimensional conformal radiotherapy (3DCRT), intensity-modulated radiotherapy (IMRT), stereotactic radiotherapy, image-guided radiotherapy (IGT), and particle therapy have facilitated the planning procedures, accurate tumor delineation, and dose estimation for effective personalized treatment. In this review, we present the technological advancements in various types of EBRT methods and discuss their clinical utility and associated limitations. We also reveal novel approaches of using biocompatible yttrium oxide scintillator-photosensitizer complex (YSM) that can generate X-ray induced cytotoxic reactive oxygen species, facilitating X-ray activated photodynamic therapy (XPDT (external beam) and/or iXPDT (internal X-ray source)) and azido-derivatives of 2-deoxy-D-glucose (2-DG) as agents for site-specific radiation-induced DNA damage.

Mechanism of reactive oxygen species generation and oxidative DNA damage induced by acrylohydroxamic acid, a putative metabolite of acrylamide

Acrylamide is formed during the heating of food and is also found in cigarette smoke. It is classified by the International Agency for Research on Cancer as a probable human carcinogen (Group 2A). Glycidamide, an epoxide metabolite of acrylamide, is implicated in the mechanism of acrylamide carcinogenicity. Acrylamide causes oxidative DNA damage in target organs. We sought to clarify the mechanism of acrylamide-induced oxidative DNA damage by investigating site-specific DNA damage and reactive oxygen species (ROS) generation by a putative metabolite of acrylamide, acrylohydroxamic acid (AA). Our results, using 32P-5′-end-labeled DNA fragments, indicated that, although AA alone did not damage DNA, AA treated with amidase induced DNA damage in the presence of Cu(II). DNA cleavage occurred preferentially at T and C, and particularly at T in 5′-TG-3′ sequences, and the DNA cleavage pattern was similar to that of hydroxylamine. The DNA damage was inhibited by methional, catalase, and Cu(I)-chelator bathocuproine, suggesting that H2O2 and Cu(I) are involved in the mechanism of DNA damage induced by AA treated with amidase. In addition, amidase-treated AA increased 8-oxo-7,8-dihydro-2′-deoxyguanosine formation in calf thymus DNA, an indicator of oxidative DNA damage, in a dose-dependent manner. In conclusion, hydroxylamine, possibly produced from AA treated with amidase, was autoxidized via the Cu(II)/Cu(I) redox cycle and H2O2 generation, suggesting that oxidative DNA damage induced by ROS plays an important role in acrylamide-related carcinogenesis.

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage.

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Autophagy restricts mitochondrial DNA damage-induced release of ENDOG (endonuclease G) to regulate genome stability

ABSTRACT Genotoxic insult causes nuclear and mitochondrial DNA damages with macroautophagy/autophagy induction. The role of mitochondrial DNA (mtDNA) damage in the requirement of autophagy for nuclear DNA (nDNA) stability is unclear. Using site-specific DNA damage approaches, we show that specific nDNA damage alone does not require autophagy for repair unless in the presence of mtDNA damage. We provide evidence that after IR exposure-induced mtDNA and nDNA damages, autophagy suppression causes non-apoptotic mitochondrial permeability, by which mitochondrial ENDOG (endonuclease G) is released and translocated to nuclei to sustain nDNA damage in a TET (tet methylcytosine dioxygenase)-dependent manner. Furthermore, blocking lysosome function is sufficient to increase the amount of mtDNA leakage to the cytosol, accompanied by ENDOG-free mitochondrial puncta formation with concurrent ENDOG nuclear accumulation. We proposed that autophagy eliminates the mitochondria specified by mtDNA damage-driven mitochondrial permeability to prevent ENDOG-mediated genome instability. Finally, we showed that HBx, a hepatitis B viral protein capable of suppressing autophagy, also causes mitochondrial permeability-dependent ENDOG mis-localization in nuclei and is linked to hepatitis B virus (HBV)-mediated hepatocellular carcinoma development. Abbreviations: 3-MA: 3-methyladenine; 5-hmC: 5-hydroxymethylcytosine; ACTB: actin beta; ATG5: autophagy related 5; ATM: ATM serine/threonine kinase; DFFB/CAD: DNA fragmentation factor subunit beta; cmtDNA: cytosolic mitochondrial DNA; ConA: concanamycin A; CQ: chloroquine; CsA: cyclosporin A; Dox: doxycycline; DSB: double-strand break; ENDOG: endonuclease G; GFP: green fluorescent protein; Gy: gray; H2AX: H2A.X variant histone; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HCC: hepatocellular carcinoma; I-PpoI: intron-encoded endonuclease; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOMP: mitochondrial outer membrane permeability; mPTP: mitochondrial permeability transition pore; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; 4-OHT: 4-hydroxytamoxifen; rDNA: ribosomal DNA; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TET: tet methylcytosine dioxygenase; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens.

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.

Quantifying site-specific chromatin mechanics and DNA damage response

DNA double-strand breaks pose a direct threat to genomic stability. Studies of DNA damage and chromatin dynamics have yielded opposing results that support either increased or decreased chromatin motion after damage. In this study, we independently measure the dynamics of transcriptionally active or repressed chromatin regions using particle tracking microrheology. We find that the baseline motion of transcriptionally repressed regions of chromatin are significantly less mobile than transcriptionally active chromatin, which is statistically similar to the bulk motion of chromatin within the nucleus. Site specific DNA damage using KillerRed tags induced in loci within repressed chromatin causes an increased motion, while loci within transcriptionally active regions remains unchanged at similar time scales. We also observe a time-dependent response associated with a further increase in chromatin decondensation. Global induction of damage with bleocin displays similar trends of chromatin decondensation and increased mobility only at 53BP1-labeled damage sites but not at non-damaged sites, indicating that chromatin dynamics are tightly regulated locally after damage. These results shed light on the evolution of the local and global DNA damage response associated with chromatin remodeling and dynamics, with direct implications for their role in repair.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

Abstract B39: Genomic copy number alterations introduce a gene-independent viability bias in CRISPR-Cas9 knock-out screens of cancer cell lines

Abstract Recent studies have demonstrated the power of CRISPR-Cas9 screening methods for identifying genetic vulnerabilities in cancer cells. As part of a larger effort to generate a comprehensive catalog of vulnerabilities, we performed CRISPR-Cas9 genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation and survival. We found a strong correlation between gene copy number and cell viability after Cas9-targeting. Copy number alterations are extremely prevalent in human cancers and frequently lead to overexpression of driver oncogenes and potential vulnerabilities. Therefore, we sought to identify such genes by investigating the relationship of genomic copy number with essentiality from our screening data. As expected, known oncogenes scored as essential in cell lines harboring amplifications of these genes. However, the scores of all other genes in these amplified regions were also strongly enriched for apparent essentiality, even among unexpressed genes. Furthermore, the infection of cells with sgRNAs targeting Cas9 to non-coding intergenic sequences within regions of high copy number gain also induced this negative effect on cell viability. We observed this effect across multiple different chromosomal structural alterations, including tandem duplications, breakage-fusion-bridge structures, and arm-level gains. More broadly, we found a striking global correlation between cell viability in response to Cas9-targeting and the genomic copy number of the targeted site, even among low-level copy number gain and loss. For example, Cas9-targeting of genes with two copies resulted in, on average, decreased viability relative to Cas9-targeting of genes with only one copy. By examining sgRNAs that target multiple genomic sites, but not within any amplified loci, we found that this cell response to Cas9-targeting correlated strongly with the total number of target sites. Together, these observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response with a severity proportional to the total number of discrete genomic loci targeted. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions. This result illustrates the sensitivity of cancer cells to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Targeting non-essential genes or non-coding intergenic sequences within regions of copy number amplification may reveal cancer-specific vulnerabilities. Citation Format: Robin M. Meyers, Andrew J. Aguirre, Barbara A. Weir, Francisca Vazquez, Cheng-Zhong Zhang, Uri Ben-David, April Cook, Gavin Ha, William F. Harrington, Mihir Doshi, Stanley Gill, Han Xu, Levi D. Ali, Guozhi Jiang, Sasha Pantel, Yenarae Lee, Amy Goodale, Andrew D. Cherniack, Coyin Oh, Gregory Kryukov, Glenn S. Cowley, Levi A. Garraway, Kimberly Stegmaier, Charles W. Roberts, Todd R. Golub, Matthew Meyerson, David E. Root, Aviad Tsherniak, William C. Hahn. Genomic copy number alterations introduce a gene-independent viability bias in CRISPR-Cas9 knock-out screens of cancer cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B39.

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803.

Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

Triplex-forming oligonucleotides targeting c-MYC potentiate the anti-tumor activity of gemcitabine in a mouse model of human cancer

Antimetabolite chemotherapy remains an essential cancer treatment modality, but often produces only marginal benefit due to the lack of tumor specificity, the development of drug resistance, and the refractoriness of slowly proliferating cells in solid tumors. Here, we report a novel strategy to circumvent the proliferation-dependence of traditional antimetabolite-based therapies. Triplex-forming oligonucleotides (TFOs) were used to target site-specific DNA damage to the human c-MYC oncogene, thereby inducing replication-independent, unscheduled DNA repair synthesis (UDS) preferentially in the TFO-targeted region. The TFO-directed UDS facilitated incorporation of the antimetabolite, gemcitabine (GEM), into the damaged oncogene, thereby potentiating the anti-tumor activity of GEM. Mice bearing COLO 320DM human colon cancer xenografts (containing amplified c-MYC) were treated with a TFO targeted to c-MYC in combination with GEM. Tumor growth inhibition produced by the combination was significantly greater than with either TFO or GEM alone. Specific TFO binding to the genomic c-MYC gene was demonstrated, and TFO-induced DNA damage was confirmed by NBS1 accumulation, supporting a mechanism of enhanced efficacy of GEM via TFO-targeted DNA damage-induced UDS. Thus, coupling antimetabolite chemotherapeutics with a strategy that facilitates selective targeting of cells containing amplification of cancer-relevant genes can improve their activity against solid tumors, while possibly minimizing host toxicity.

Serial analysis of mutation spectra (SAMS): a new approach for the determination of mutation spectra of site-specific DNA damage and their sequence dependence

Many mutations occur as a result of DNA synthesis past the site of DNA damage by DNA damage bypass polymerases. The frequency and types of mutations not only depend on the nature of the damage, but also on the sequence context, as revealed from analysis of mutation spectra of DNA exposed to mutagens. Herein we report a new method for the rapid determination of the effect of sequence context on mutagenesis called SAMS for serial analysis of mutation spectra. This technique makes use of the methodology that underlies serial analysis of gene expression (SAGE) to analyze mutations that result from DNA synthesis past a DNA lesion site-specifically embedded in a library of DNA sequences. To illustrate our technique we determined the effect of sequence context on mutations generated by DNA synthesis past a tetrahydrofuran abasic site model by the DNA damage bypass polymerase yeast polymerase η.

Mechanism of DNA Damage and Apoptosis Induced by Anticancer Drugs through Generation of Reactive Oxygen Species

A number of anticancer drugs exert their effect by causing DNA damage and subsequent apoptosis induction. Reactive oxygen species (ROS), such as hydrogen peroxide (H(2)O(2)) and super oxide anion (O(2)(-)), participate in apoptosis and DNA damage induced by some anticancer drugs, however, the precise mechanism of apoptosis via ROS formation remains to be clarified. I investigated the mechanism of apoptosis and DNA damage induced by anticancer drugs, especially topoisomerase inhibitors, using human cultured cells. TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by poly (ADP-ribose) polymerase (PARP) and NAD(P)H oxidase activation. Doxorubicin (DOX), an anthracycline antibiotic and topoisomerase inhibitor, induces apoptosis through direct oxidative DNA damage leading to indirect H(2)O(2) generation mediated by PARP and NAD(P)H oxidase activation. DOX caused site-specific oxidative DNA damage in the presence of copper(II), which may contribute to apoptosis. These findings suggest that ROS formation plays important roles in apoptosis induced by anticancer drugs. Furthermore, these studies may provide an insight into the development of new effective chemotherapeutic drugs.

Mechanism of site-specific DNA damage induced by ozone

Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2′-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline- N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D 2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D 2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.