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DNA Repair Mar 2009Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The...
Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.
Topics: Alkylating Agents; Animals; Cattle; Cell Survival; Cerebellum; Chickens; DNA; DNA Fragmentation; DNA Glycosylases; DNA Modification Methylases; DNA Repair; DNA Repair Enzymes; Ethylamines; Humans; Mechlorethamine; Methylazoxymethanol Acetate; Mice; Motor Activity; Neurons; Sulfuric Acid Esters; Tumor Suppressor Proteins
PubMed: 19162564
DOI: 10.1016/j.dnarep.2008.12.002 -
Nucleic Acids Research Dec 1998Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking...
Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences. In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.
Topics: Alkylating Agents; Alkylation; Animals; Cross-Linking Reagents; DNA Adducts; DNA Footprinting; DNA Fragmentation; Guanine; Hot Temperature; Humans; Models, Chemical; Monocrotaline; Mutagenesis; Plasmids
PubMed: 9826770
DOI: 10.1093/nar/26.23.5441 -
Molecular and Cellular Biology Sep 1991The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a...
The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts. The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism. We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA. These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells. Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment. The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1. An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells. The rate of spontaneous mutation in apn1 mutant S. cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells. This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.
Topics: Alkylating Agents; Alkylation; Centrifugation, Density Gradient; DNA Damage; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Directed DNA Polymerase; Deoxyribonuclease IV (Phage T4-Induced); Endodeoxyribonucleases; Escherichia coli Proteins; Genes, Fungal; Hydrogen Peroxide; Methyl Methanesulfonate; Mutation; Oxidation-Reduction; Saccharomyces cerevisiae
PubMed: 1715020
DOI: 10.1128/mcb.11.9.4537-4544.1991 -
DNA Repair Mar 2013Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While...
Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.
Topics: Alkylating Agents; Base Sequence; Binding, Competitive; Cytoplasm; DNA Breaks, Single-Stranded; DNA Glycosylases; DNA Repair; DNA, Single-Stranded; DNA-Binding Proteins; HEK293 Cells; HeLa Cells; Humans; Methyl Methanesulfonate; Mitochondria; Mitochondrial Proteins; Protein Binding; Protein Transport
PubMed: 23290262
DOI: 10.1016/j.dnarep.2012.11.009 -
Mutation Research. Genetic Toxicology... Apr 2019DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer...
DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.
Topics: Acetaldehyde; Alkylating Agents; DNA Damage; DNA, Bacterial; Epoxy Compounds; Escherichia coli; Esterases; Rec A Recombinases; SOS Response, Genetics
PubMed: 30857727
DOI: 10.1016/j.mrgentox.2019.02.001 -
The Journal of Biological Chemistry Dec 2014Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is...
Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.
Topics: Alkylation; Carrier Proteins; DNA Damage; DNA Glycosylases; DNA Methylation; DNA Repair; DNA, Fungal; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 25381260
DOI: 10.1074/jbc.M114.590216 -
Journal of Cellular and Molecular... Oct 2016The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1,...
The SN 1 alkylating agents activate the mismatch repair system leading to delayed G2 /M cell cycle arrest and DNA repair with subsequent survival or cell death. STAT1, an anti-proliferative and pro-apoptotic transcription factor is known to potentiate p53 and to affect DNA-damage cellular response. We studied whether STAT1 may modulate cell fate following activation of the mismatch repair system upon exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Using STAT1-proficient or -deficient cell lines, we found that STAT1 is required for: (i) reduction in the extent of DNA lesions, (ii) rapid phosphorylation of T68-CHK2 and of S15-p53, (iii) progression through the G2 /M checkpoint and (iv) long-term survival following treatment with MNNG. Presence of STAT1 is critical for the formation of a p53-DNA complex comprising: STAT1, c-Abl and MLH1 following exposure to MNNG. Importantly, presence of STAT1 allows recruitment of c-Abl to p53-DNA complex and links c-Abl tyrosine kinase activity to MNNG-toxicity. Thus, our data highlight the important modulatory role of STAT1 in the signalling pathway activated by the mismatch repair system. This ability of STAT1 to favour resistance to MNNG indicates the targeting of STAT1 pathway as a therapeutic option for enhancing the efficacy of SN1 alkylating agent-based chemotherapy.
Topics: Alkylating Agents; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Survival; Checkpoint Kinase 2; Cytoprotection; DNA; DNA Breaks, Double-Stranded; Histones; Humans; Imatinib Mesylate; Methylnitronitrosoguanidine; Protein Isoforms; Proto-Oncogene Proteins c-abl; STAT1 Transcription Factor; Tumor Suppressor Protein p53
PubMed: 27464833
DOI: 10.1111/jcmm.12887 -
Nucleic Acids Research Aug 2012The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The...
The Escherichia coli AlkB protein (EcAlkB) is a DNA repair enzyme which reverses methylation damage such as 1-methyladenine (1-meA) and 3-methylcytosine (3-meC). The mammalian AlkB homologues ALKBH2 and ALKBH3 display EcAlkB-like repair activity in vitro, but their substrate specificities are different, and ALKBH2 is the main DNA repair enzyme for 1-meA in vivo. The genome of the model plant Arabidopsis thaliana encodes several AlkB homologues, including the yet uncharacterized protein AT2G22260, which displays sequence similarity to both ALKBH2 and ALKBH3. We have here characterized protein AT2G22260, by us denoted ALKBH2, as both our functional studies and bioinformatics analysis suggest it to be an orthologue of mammalian ALKBH2. The Arabidopsis ALKBH2 protein displayed in vitro repair activities towards methyl and etheno adducts in DNA, and was able to complement corresponding repair deficiencies of the E. coli alkB mutant. Interestingly, alkbh2 knock-out plants were sensitive to the methylating agent methylmethanesulphonate (MMS), and seedlings from these plants developed abnormally when grown in the presence of MMS. The present study establishes ALKBH2 as an important enzyme for protecting Arabidopsis against methylation damage in DNA, and suggests its homologues in other plants to have a similar function.
Topics: AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alkylating Agents; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; DNA Damage; DNA Methylation; DNA Repair; Dioxygenases; Escherichia coli Proteins; Genome, Plant; Methyl Methanesulfonate; Mixed Function Oxygenases; Molecular Sequence Data; Sequence Alignment
PubMed: 22532610
DOI: 10.1093/nar/gks327 -
The Journal of Biological Chemistry Nov 2002Alkylating agents are generally highly reactive with DNA but demonstrate limited DNA sequence selectivity. In contrast, synthetic pyrrole-imidazole polyamides recognize...
Alkylating agents are generally highly reactive with DNA but demonstrate limited DNA sequence selectivity. In contrast, synthetic pyrrole-imidazole polyamides recognize specific DNA sequences with high affinity but are unable to permanently damage DNA. An eight-ring hairpin polyamide conjugated to the alkylating moiety cyclopropylpyrroloindole, related to the natural product CC-1065, affords a conjugate 1-CBI (polyamide 1-CBI (1-(chloromethyl)-5-hydroxyl-1,2-dihydro-3H-benz[e]indole) conjugate), which binds to specific sequences in the minor groove of DNA and alkylates a single adenine flanking the polyamide binding site. In this study, we show that 1-CBI alkylates DNA in both plasmid and intracellular minichromosomal form and inhibits DNA replication under both cell-free and cellular conditions. In addition, it inhibits cell growth and arrests cells in the G2/M phase of the cell cycle.
Topics: Alkylating Agents; Base Sequence; Breast Neoplasms; Cell Cycle; Cell Division; DNA; DNA Damage; DNA, Viral; Female; Humans; Indoles; Nucleic Acid Conformation; Nylons; Oligodeoxyribonucleotides; Pyrroles; Simian virus 40; Tumor Cells, Cultured
PubMed: 12196541
DOI: 10.1074/jbc.M207179200 -
Journal of Neuro-oncology Jan 2024Malignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy...
BACKGROUND
Malignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ.
METHODS
Via lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis.
RESULTS
We used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects.
CONCLUSION
dCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications.
Topics: Humans; Antineoplastic Agents, Alkylating; Brain Neoplasms; Dacarbazine; DNA; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; Glioma; Guanine; O(6)-Methylguanine-DNA Methyltransferase; Temozolomide
PubMed: 38224404
DOI: 10.1007/s11060-023-04531-z