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Mutation Research. Genetic Toxicology... 2022Cells have developed diverse protective mechanisms that enable them to tolerate low doses of genotoxic compounds. DNA repair processes attenuate the mutagenic and...
Cells have developed diverse protective mechanisms that enable them to tolerate low doses of genotoxic compounds. DNA repair processes attenuate the mutagenic and carcinogenic effects of alkylating agents, and multiple studies indicate a key role of specific DNA repair factors and pathways in establishing non-linear dose response relationships. Using an overexpression approach, we investigated the impact of O-methylguanine-DNA-methyltransferase (MGMT), which repairs O-methylguanine (OMeG) in a damage reversal reaction, and N-methylpurine-DNA glycosylase (MPG), which acts as an apical enzyme in the BER pathway, on the DNA damage response to the alkylating agents MNNG and MMS. Our data indicate a clear protective effect of MGMT against MNNG-induced nuclear γH2AX foci formation, sister chromatid exchanges (SCE) and cytotoxicity, as determined in the colony formation assay. MGMT protected with similar efficiency against MMS-induced cytotoxicity and γH2AX foci formation, but suppressed SCE induction only weakly, which indicates that recombination events induced by MMS result from other lesions than OMeG. In contrast, overexpression of MPG had only a very mild protective effect on the cellular defense against MMS and MNNG. Collectively, our data indicate that overexpression of MGMT results in non-linear DNA damage responses to OMeG inducers. In contrast, MPG overexpression has only minor impact on the DNA damage response to alkylating drugs, indicating that other downstream enzymes in the BER pathway are limiting.
Topics: Alkylating Agents; DNA Repair; Methylnitronitrosoguanidine; O(6)-Methylguanine-DNA Methyltransferase; Sister Chromatid Exchange
PubMed: 35483785
DOI: 10.1016/j.mrgentox.2022.503462 -
Molecular Cell Sep 2009DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA...
DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.
Topics: Alkylating Agents; Animals; Ataxia Telangiectasia Mutated Proteins; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Checkpoint Kinase 1; DNA; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Fanconi Anemia Complementation Group A Protein; Fanconi Anemia Complementation Group D2 Protein; HeLa Cells; Humans; Nucleic Acid Conformation; Protein Kinases; Protein Serine-Threonine Kinases; Recombinant Proteins; Replication Origin; Replication Protein A; Signal Transduction; Time Factors; Transfection; Xenopus Proteins; Xenopus laevis
PubMed: 19748363
DOI: 10.1016/j.molcel.2009.08.014 -
Chembiochem : a European Journal of... Jan 2017Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin...
Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin imidazole-pyrrole polyamide 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-CBI) conjugates that produce sequence-specific DNA ICLs were designed and synthesized. Conjugates with H-pin polyamide and seco-CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high-resolution denaturing gel electrophoresis and sequence-specific plasmid cleavage. One conjugate (6), which contained a chiral (S)-seco-CBI, exhibited greater sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)-CBI unit.
Topics: Alkylation; Antineoplastic Agents, Alkylating; Base Sequence; Cell Line, Tumor; Cell Survival; DNA; Electrophoresis, Polyacrylamide Gel; Humans; Imidazoles; Indoles; Intercalating Agents; Plasmids; Pyrroles; Stereoisomerism
PubMed: 27862755
DOI: 10.1002/cbic.201600425 -
Chemical Research in Toxicology Apr 2017Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all...
Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all cancer cases, and novel platinum based agents are under development. The antitumor effects of cisplatin and other platinum compounds are attributed to their ability to induce interstrand DNA-DNA cross-links, which are thought to inhibit tumor cell growth by blocking DNA replication and/or preventing transcription. However, platinum agents also induce significant numbers of unusually bulky and helix-distorting DNA-protein cross-links (DPCs), which are poorly characterized because of their unusual complexity. We and others have previously shown that DPCs block DNA replication and transcription and causes toxicity in human cells, potentially contributing to the biological effects of platinum agents. In the present work, we have undertaken a system-wide investigation of cisplatin-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells using mass spectrometry-based proteomics. DPCs were isolated from cisplatin-treated cells using a modified phenol/chloroform DNA extraction in the presence of protease inhibitors. Proteins were released from DNA strands and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins captured on chromosomal DNA following treatment with cisplatin were identified, including high mobility group (HMG) proteins, histone proteins, and elongation factors. To reveal the exact molecular structures of cisplatin-mediated DPCs, isotope dilution HPLC-ESI-MS/MS was employed to detect 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Our results demonstrate that therapeutic levels of cisplatin induce a wide range of DPC lesions, which likely contribute to both target and off target effects of this clinically important drug.
Topics: Antineoplastic Agents, Alkylating; Blotting, Western; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cisplatin; DNA; DNA Replication; Fibrosarcoma; Guanine; Humans; Peptides; Proteins; Proteomics; Tandem Mass Spectrometry
PubMed: 28282121
DOI: 10.1021/acs.chemrestox.6b00389 -
Molecular Cancer Research : MCR Dec 2012To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma-derived...
To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma-derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored toward "druggable" targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases uracil-DNA glycosylase (UNG) and A/G-specific adenine DNA glycosylase (MYH) as well as methylpurine-DNA glycosylase (MPG) to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in Escherichia coli and Saccharomyces cerevisiae. The conserved biologic processes across all three species compose an alkylation functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance.
Topics: Alkylation; Antineoplastic Agents, Alkylating; Bacterial Proteins; Cell Line, Tumor; DNA Damage; DNA Glycosylases; DNA Repair; Dacarbazine; Drug Resistance, Neoplasm; Escherichia coli; Glioblastoma; Humans; N-Glycosyl Hydrolases; Reactive Oxygen Species; Saccharomyces cerevisiae; Temozolomide; Ubiquitin-Protein Ligases; Uracil-DNA Glycosidase
PubMed: 23038810
DOI: 10.1158/1541-7786.MCR-12-0168 -
The Biochemical Journal Jan 1975Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-,...
Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.
Topics: 1-Propanol; Adenine; Alkylation; DNA; Ethanol; Guanine; Mass Spectrometry; Mesylates; Purines
PubMed: 172066
DOI: 10.1042/bj1450073 -
Journal of Bacteriology Jun 1994The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring...
The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.
Topics: Alkylating Agents; Alkylation; Bacterial Proteins; Cell Division; DNA Modification Methylases; DNA Repair; Escherichia coli; Escherichia coli Proteins; Lac Operon; Methyltransferases; Mutation; O(6)-Methylguanine-DNA Methyltransferase; Transcription Factors
PubMed: 8195077
DOI: 10.1128/jb.176.11.3224-3230.1994 -
The Biochemical Journal Jul 1994The one- and two-electron enzymic reduction of the bioreductive alkylating agents 2-methylmethoxynaphthoquinone (quinone I) and 2-chloromethylnaphthoquinone (quinone II)...
One- and two-electron reduction of 2-methyl-1,4-naphthoquinone bioreductive alkylating agents: kinetic studies, free-radical production, thiol oxidation and DNA-strand-break formation.
The one- and two-electron enzymic reduction of the bioreductive alkylating agents 2-methylmethoxynaphthoquinone (quinone I) and 2-chloromethylnaphthoquinone (quinone II) was studied with purified NADPH-cytochrome P-450 reductase and DT-diaphorase respectively, and characterized in terms of kinetic constants, oxyradical production, thiol oxidation and DNA-strand-break formation. The catalytic-centre activity values indicated that DT-diaphorase catalysed the reduction of quinone I far more efficiently than NADPH-cytochrome P-450 reductase, although the Km values of the two enzymes for this quinone were similar (1.2-3.0 microM). The one-electron-transfer flavoenzyme also catalysed the reduction of quinone II, but the behaviour of DT-diaphorase towards this quinone did not permit calculation of kinetic constants. A salient feature of the redox transitions caused by the one- and two-electron catalysis of these quinones was the different contributions of disproportionation and autoxidation reactions respectively. In the former case, about 26% of NADPH consumed was accounted for in terms of autoxidation (as H2O2 formation), whereas in the latter, the autoxidation component accounted for most (98%) of the NADPH consumed. This difference was abrogated by superoxide dismutase, which enhanced autoxidation during NADPH-cytochrome P-450 catalysis to a maximal value. E.s.r. analysis indicated the formation of superoxide radicals, the signal of which was suppressed by superoxide dismutase and unaffected by catalase. The one- and two-electron reduction of these quinones in the presence of GSH was accompanied by formation of thiyl radicals. Although superoxide dismutase suppressed the thiol radical e.s.r. signal in both instances, the enzyme enhanced GSSG accumulation during NADPH-cytochrome P-450 catalysis of quinone I, whereas it inhibited GSSG formation during reduction of the quinone by DT-diaphorase. One- and two-electron reduction of quinone I led to calf thymus DNA-strand-break formation, a process that (a) was substantially decreased in experiments performed with dialysed DNA and in the presence of desferal and (b) was partially sensitive to superoxide dismutase and/or catalase. These findings are rationalized in terms of the occurrence of metal ions ligated to DNA, protecting against the toxic effects of superoxide radicals generated during enzymic reduction of quinones.
Topics: Alkylating Agents; Animals; Cattle; DNA; DNA Damage; Electrons; Free Radicals; Glutathione; In Vitro Techniques; Kinetics; NAD(P)H Dehydrogenase (Quinone); NADPH-Ferrihemoprotein Reductase; Naphthoquinones; Oxidation-Reduction; Sulfhydryl Compounds
PubMed: 8037673
DOI: 10.1042/bj3010021 -
The Biochemical Journal Oct 2003AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from...
AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.
Topics: Alkylating Agents; Archaeoglobus fulgidus; Bacteria; DNA; DNA Damage; DNA Repair; Enzyme Stability; Guanine; Hydrogen-Ion Concentration; O(6)-Methylguanine-DNA Methyltransferase; Protein Folding; Protein Renaturation; Temperature
PubMed: 12892560
DOI: 10.1042/BJ20030809 -
FEBS Letters Oct 1975
Review
Topics: Alkylating Agents; Animals; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Binding Sites; Cell Division; Cisplatin; DNA; Daunorubicin; Doxorubicin; Humans; Leukemia L1210; Mice; Microsomes, Liver; Neoplasms; Structure-Activity Relationship
PubMed: 1102331
DOI: 10.1016/0014-5793(75)80305-x