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Chemical Research in Toxicology Apr 2020Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the...
Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The monoalkylated N7-guanine (NM-G) adduct and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened -substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM-Fapy-G adducts in liver, lung, and spleen over 168 h. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three-times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 h post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing . The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.
Topics: Animals; DNA Adducts; Female; Kinetics; Mass Spectrometry; Mechlorethamine; Mice; Mice, Inbred C57BL; Molecular Structure; Tissue Distribution
PubMed: 32174110
DOI: 10.1021/acs.chemrestox.0c00012 -
Plant Physiology May 2020Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley () and is involved in the plant immune response dependent on jasmonate...
Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley () and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in , as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.
Topics: Catalytic Domain; Hordeum; Plant Leaves; Plant Proteins; Ribosomes; Nicotiana
PubMed: 32123042
DOI: 10.1104/pp.19.01029 -
Chemical Science Dec 2019DNA-encoded compound libraries are a widely used technology for target-based small molecule screening. Generally, these libraries are synthesized by solution phase...
DNA-encoded compound libraries are a widely used technology for target-based small molecule screening. Generally, these libraries are synthesized by solution phase combinatorial chemistry requiring aqueous solvent mixtures and reactions that are orthogonal to DNA reactivity. Initiating library synthesis with readily available controlled pore glass-coupled DNA barcodes benefits from enhanced DNA stability due to nucleobase protection and choice of dry organic solvents for encoded compound synthesis. We screened the compatibility of solid-phase coupled DNA sequences with 53 metal salts and organic reagents. This screening experiment suggests design of encoded library synthesis. Here, we show the reaction optimization and scope of three sp-bond containing heterocyclic scaffolds synthesized on controlled pore glass-connected DNA sequences. A ZnCl-promoted aza-Diels-Alder reaction with Danishefsky's diene furnished diverse substituted DNA-tagged pyridones, and a phosphoric acid organocatalyst allowed for synthesis of tetrahydroquinolines by the Povarov reaction and pyrimidinones by the Biginelli reaction, respectively. These three reactions caused low levels of DNA depurination and cover broad and only partially overlapping chemical space though using one set of DNA-coupled starting materials.
PubMed: 32055372
DOI: 10.1039/c9sc04708e -
Biochemistry Feb 2020Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA...
Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.
Topics: Cross-Linking Reagents; DNA; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA Repair; Deoxyribonuclease IV (Phage T4-Induced); Escherichia coli; Peptides; Plasmids; Polyketides
PubMed: 31977191
DOI: 10.1021/acs.biochem.9b01070 -
Electrophoresis Mar 2020The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results,...
The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical-chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.
Topics: DNA, Complementary; Forensic Genetics; Humans; Polymerase Chain Reaction; RNA; RNA Stability; Sensitivity and Specificity
PubMed: 31967656
DOI: 10.1002/elps.201900200 -
Molecules (Basel, Switzerland) Oct 2019A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant...
A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.
Topics: Alkylation; Base Pairing; Catalytic Domain; DNA; DNA Damage; DNA-Directed DNA Polymerase; Deoxycytosine Nucleotides; Deoxyguanosine; Humans; Kinetics; Metals; Models, Molecular; Nucleic Acid Conformation
PubMed: 31683505
DOI: 10.3390/molecules24213928 -
Analytical Chemistry Dec 2019DNA-DNA crosslinks, especially interstrand crosslinks (ICLs), cause cytotoxicity via blocking replication and transcription. Most measurements of ICLs lack sensitivity...
DNA-DNA crosslinks, especially interstrand crosslinks (ICLs), cause cytotoxicity via blocking replication and transcription. Most measurements of ICLs lack sensitivity and structural information. Here, a high resolution, accurate mass spectrometry (HRMS) method was developed to comprehensively determine the untargeted, totality of DNA crosslinks, a.k.a. DNA crosslinkomics. Two novel features were introduced into this method: the accurate mass neutral losses of both two 2-deoxyribose (dR) and one dR groups will screen for ICLs as modified dinucleosides; the accurate mass neutral losses of both of the two nucleobases and one nucleobase will detect unstable DNA crosslinks, that could undergo depurination. Our crosslinkomics approach was tested by screening for crosslinks in formaldehyde- and chlorambucil-treated calf thymus DNA. The results showed that all expected drug-bridged crosslinks were detected successfully, along with various unexpected crosslinks. Using HRMS, the molecular formula and chemical structures of these unexpected crosslinks were determined. The formation of apurinic/apyrimidinic (AP) site-derived crosslinks, at levels comparable to those for drug-bridged crosslinks, highlighted their novel, potential role in cytotoxicity. Our new crosslinkomics approach can detect expected and unexpected environmental and drug-induced crosslinks in biological samples. This broadens the existing cellular DNA adductome and offers the potential to become a powerful tool in precision medicine.
Topics: Animals; Cattle; Chlorambucil; Chromatography, Liquid; Cross-Linking Reagents; DNA; Formaldehyde; Mass Spectrometry
PubMed: 31670503
DOI: 10.1021/acs.analchem.9b04068 -
The Journal of Biological Chemistry Nov 2019Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA...
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of ( for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of mRNA. We show that mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes mRNA that is associated with ribosomes.
Topics: Basic-Leucine Zipper Transcription Factors; Endoplasmic Reticulum Stress; Gene Expression Regulation, Fungal; Membrane Glycoproteins; Models, Biological; Mutation; Protein Multimerization; Protein Serine-Threonine Kinases; Purines; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Messenger; Repressor Proteins; Ribosomes; Ricin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Unfolded Protein Response
PubMed: 31624149
DOI: 10.1074/jbc.RA119.009128 -
Chemical Research in Toxicology Oct 2019N3-Methyl-2'-deoxyadenosine (MdA) is the major dA methylation product in duplex DNA. MdA blocks DNA replication and undergoes depurination at significantly higher rates...
N3-Methyl-2'-deoxyadenosine (MdA) is the major dA methylation product in duplex DNA. MdA blocks DNA replication and undergoes depurination at significantly higher rates than the native nucleotide from which it is derived. Recent reports on the effects of the nucleosome core particle (NCP) environment on the reactivity of N7-methyl-2'-deoxyguanosine (MdG) inspired this investigation concerning the reactivity of MdA in NCPs. NCPs containing MdA at selected positions were produced using a strategy in which the minor groove binding Me-Lex molecule serves as a sequence specific methylating agent. Hydrolysis of the glycosidic bond in MdA to form abasic sites (AP) is suppressed in a NCP. Experiments using histone variants indicate that the proximal, highly basic N-terminal tails are partially responsible for the decreased depurination rate constant. MdA also forms cross-links with histone proteins. The levels of MdA-histone DNA-protein cross-links (DPC) decrease significantly over time and are replaced by those involving AP. The time dependent decrease in DPC is attributed to the reversibility of their formation and the relatively rapid rate of AP formation from MdA. Overall, MdA reactivity in NCPs qualitatively resembles that of MdG.
Topics: DNA; DNA-Binding Proteins; Deoxyadenosines; Nucleic Acid Conformation; Nucleosomes
PubMed: 31565933
DOI: 10.1021/acs.chemrestox.9b00299 -
Bioscience Reports Oct 2019Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the...
Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.
Topics: Binding Sites; Hydrophobic and Hydrophilic Interactions; Leucine; Ribosomes; Ricin; Saccharomyces cerevisiae
PubMed: 31548364
DOI: 10.1042/BSR20192022