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PloS One 2014Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been...
Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been implicated in a variety of diseases, including cancer. Here, we demonstrate a new approach for the detection of depurination at the single-molecule scale using solid-state nanopores. We induce depurination in short duplex DNA using acidic conditions and observe that the presence of apurinic sites results in significantly slower dynamics during electrokinetic translocation. This procedure may be valuable as a diagnostic for in situ quantification of DNA depurination.
Topics: Apurinic Acid; Base Sequence; Biosensing Techniques; DNA; Humans; Molecular Sequence Data; Nanopores; Purines
PubMed: 24988437
DOI: 10.1371/journal.pone.0101632 -
FEBS Letters Oct 1990Mammalian ribosomes have been shown to be enzymatically modified by ribosomal inactivating protein (RIPs) via specific depurination of rRNA. Here we report that...
Mammalian ribosomes have been shown to be enzymatically modified by ribosomal inactivating protein (RIPs) via specific depurination of rRNA. Here we report that ribosomes isolated from wheat germ contain intact and undepurinated rRNA and are depurinated by pokeweed antiviral protein (PAP). Pokeweed ribosomes isolated under the same conditions are depurinated. Total RNA isolated from pokeweed in the presence of strong denaturants was found to pbe partially depurinated. We conclude that wheat germ ribosomes are resistant to the endogenous RIP, tritin, but are sensitive to PAP and that pokeweed ribosomes can be depurinated by the N-glycosidase activity of endogenous PAP during isolation.
Topics: Antiviral Agents; Base Sequence; Molecular Sequence Data; N-Glycosyl Hydrolases; Plant Proteins; Plants; Purines; RNA, Ribosomal; Ribosome Inactivating Proteins, Type 1; Ribosomes; Triticum
PubMed: 2226845
DOI: 10.1016/0014-5793(90)81070-5 -
Proceedings of the National Academy of... Mar 1984The mutagenic consequences of damage to DNA produced by low pH and high temperature have been determined in a forward mutational system capable of detecting all classes...
The mutagenic consequences of damage to DNA produced by low pH and high temperature have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When damaged single-stranded DNA from bacteriophage M13mp2 is used to transfect competent Escherichia coli cells, a 15-fold increase in mutation frequency, measured as loss of alpha-complementation by the lac DNA in the phage, is observed compared with an untreated DNA control transfection. The enhanced mutagenicity is largely dependent on induction of the error-prone SOS response and is proportional to the number of lethal hits introduced into the DNA. The effect is abolished by treatment of the damaged DNA before transfection with either apurinic/apyrimidinic endonuclease or alkali. Based on these observations and the rate constants for formation of the known heat/acid-produced lesions in DNA, it is concluded that the majority of the induced mutagenesis results from apurinic sites. DNA sequence analysis of 87 spontaneous and 124 induced mutants indicates that the major effect is on single base-substitution mutagenesis with a small increase in (deletion) frame-shift frequency. Approximately 80% of the base-substitution mutations occur at purine positions in the viral strand, consistent with depurination as the predominant premutagenic lesion. The preference of guanine over adenine sites mutated is consistent with the preference for depurination of guanine over adenine. Transversions are observed for 57 of 79 (72%) induced base substitutions, with a strong preference for insertion of adenine residues opposite the putative apurinic site. These data in a forward mutational system provide insight into the mechanisms used by a cell to replicate DNA containing noncoding lesions.
Topics: Apurinic Acid; Base Sequence; Coliphages; DNA Repair; DNA, Viral; Escherichia coli; Mutation; Polynucleotides; Templates, Genetic; Transfection
PubMed: 6369329
DOI: 10.1073/pnas.81.5.1494 -
Chemistry (Weinheim An Der Bergstrasse,... Jun 2022Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved...
Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with G and G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.
Topics: Nucleosides; Plant Proteins; RNA; RNA, Ribosomal; Ribosome Inactivating Proteins; Ribosomes
PubMed: 35390188
DOI: 10.1002/chem.202200994 -
Chemical Communications (Cambridge,... Jun 2007A deoxyribozyme is identified that mediates the site-selective depurination of its 5'-terminal guanosine nucleotide using periodate (IO(4)-) as an obligatory cofactor.
A deoxyribozyme is identified that mediates the site-selective depurination of its 5'-terminal guanosine nucleotide using periodate (IO(4)-) as an obligatory cofactor.
Topics: Amination; DNA, Catalytic; Guanosine; Models, Chemical; Oligonucleotides; Periodic Acid; Purines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 17534508
DOI: 10.1039/b704507g -
Drug Metabolism and Disposition: the... May 2019Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic...
Duocarmycins [including cyclopropyl pyrroloindole (CPI) or cyclopropyl benzoindole (CBI)] are a class of DNA minor-groove alkylators and seco-CPI/CBIs are synthetic pro-forms that can spirocyclize to CPI/CBI. Bis-CPI/CBIs are potential drug candidates because of their enhanced cytotoxicity from DNA crosslinking, but it is difficult to analyze them for structure-activity correlation because of their DNA reactivity. To study their DNA alkylation, neutral thermal hydrolysis has been frequently applied to process depurination. However, unwanted side reactions under this condition have been reported, which could lead to poor correlation of DNA alkylation data with efficacy results, especially for bis-CPI/CBIs. In this study, an acidic depurination method was developed and applied for analysis of DNA alkylation and shown to be an easier and milder method than the traditional neutral thermal hydrolysis. DNA alkylation and stability of three bis-seco-CBIs were characterized in comparison with two mono-seco-CPIs. The results suggested that: 1) The acidic depurination method was capable of capturing a more representative population, sometimes a different population, of DNA adducts as they existed on DNA compared with the heat depurination method. 2) Di-adenine adducts were captured as expected for the CBI dimers, although the major type of adduct was still mono-adenine adducts. 3) The rate of DNA alkylation, DNA adduct profile, and relative amounts of di-adduct versus mono-adduct were significantly affected by the size, and possibly lipophilicity, of the nonalkylating part of the molecules. 4) Spirocyclization and amide hydrolysis represented two major pathways of degradation. Overall, by applying acidic depurination analyses, this study has illustrated DNA adduct characteristics of novel bis-seco-CBIs with dominating mono-alkylation and provides an alternative method for evaluating DNA minor-groove alkylators. These findings provide an effective analytical tool to evaluate DNA alkylators and to study the DNA alkylation that is a disposition mechanism of these compounds.
Topics: Adenine; Alkylating Agents; Alkylation; Antineoplastic Agents, Alkylating; DNA; DNA Adducts; Duocarmycins
PubMed: 30858239
DOI: 10.1124/dmd.118.085209 -
Proceedings of the National Academy of... Mar 1981Introduction of apurinic sites into phi X174 am3 DNA leads to loss of biological activity when measured in a transfection assay. For single-stranded DNA, approximately...
Introduction of apurinic sites into phi X174 am3 DNA leads to loss of biological activity when measured in a transfection assay. For single-stranded DNA, approximately one apurinic site constitutes a lethal hit; for double-stranded (RFI) DNA, approximately 3.5 hits per strand are lethal. When the reversion frequency of am3 DNA is measured, no increase due to depurination is observed above the background level. However, a large increase in reversion frequency is observed when the same DNA is assayed by using spheroplasts derived from bacteria previously exposed to UV light. The results suggest that apurinic sites are impediments to a replicating DNA polymerase; however, nucleotides can be incorporated opposite these sites under SOS-induced conditions. We estimate the frequency of mutagenesis per apurinic site to be less than 1 in 1400 in normal spheroplasts and 1 in 100 in SOS-induced spheroplasts.
Topics: Apurinic Acid; Bacteriophage phi X 174; DNA Repair; DNA Replication; DNA, Viral; Escherichia coli; Kinetics; Mutation; Polynucleotides; Transfection; Virus Replication
PubMed: 6453349
DOI: 10.1073/pnas.78.3.1773 -
Cold Spring Harbor Perspectives in... Jul 2013Under favorable conditions DNA can survive for thousands of years in the remains of dead organisms. The DNA extracted from such remains is invariably degraded to a small... (Review)
Review
Under favorable conditions DNA can survive for thousands of years in the remains of dead organisms. The DNA extracted from such remains is invariably degraded to a small average size by processes that at least partly involve depurination. It also contains large amounts of deaminated cytosine residues that are accumulated toward the ends of the molecules, as well as several other lesions that are less well characterized.
Topics: DNA; DNA Damage; DNA Fragmentation; Deamination; Sequence Analysis, DNA; Time Factors
PubMed: 23729639
DOI: 10.1101/cshperspect.a012567 -
Biochemistry Oct 2013DNA glycosylase AlkD excises N7-methylguanine (7mG) by a unique but unknown mechanism, in which the damaged nucleotide is positioned away from the protein and the...
DNA glycosylase AlkD excises N7-methylguanine (7mG) by a unique but unknown mechanism, in which the damaged nucleotide is positioned away from the protein and the phosphate backbone is distorted. Here, we show by methylphosphonate substitution that a phosphate proximal to the lesion has a significant effect on the rate enhancement of 7mG depurination by the enzyme. Thus, instead of a conventional mechanism whereby protein side chains participate in N-glycosidic bond cleavage, AlkD remodels the DNA into an active site composed exclusively of DNA functional groups that provide the necessary chemistry to catalyze depurination.
Topics: Catalysis; Catalytic Domain; Crystallography, X-Ray; DNA; DNA Glycosylases; DNA Repair; Guanine; Humans; Models, Molecular; Protein Conformation; Purines
PubMed: 24090276
DOI: 10.1021/bi401195r -
Carcinogenesis Mar 1992Alkylation of DNA by chloroethylnitrosourea (CNU) at the guanine N7 position has been shown to occur in a sequence-selective fashion. In this report we find that the...
Alkylation of DNA by chloroethylnitrosourea (CNU) at the guanine N7 position has been shown to occur in a sequence-selective fashion. In this report we find that the depurination of these alkylated sites occurs with two distinct kinetic components--GG sequences depurinate within 30 min of exposure to CNU, while depurination at GT sequences is first observed after 1 h and continues to increase 16 h after drug exposure. These apurinic sites are converted to DNA strand breaks and constitute less than 10% of the total sites of guanine N7 alkylation. Spermidine was found to decrease alkylation in 5'-GG-3' sequences but increases alkylation at 5'-GTC-3' sequences. These findings suggest that the majority of the guanine N7 alkylations formed by CNU are stable, with a minor adduct being responsible for the slow depurination event. We propose that the rapid depurination induced by CNU occurs from an initial guanine O6 alkylation, which then depurinates via a guanine O6-N7 cyclized intermediate. We also propose that the resulting apurinic sites may lead to DNA interstrand cross-linking (ISC). In support of these hypotheses we show that (i) DNA modified with the monoalkylating agent dimethylsulfate forms DNA ISC upon depurination; (ii) ellagic acid enhances the level of guanine N7 alkylation and alters the pattern of sequence selectivity shown by three bifunctional chloroethylating agents CNU, mitozolomide and methyl 3-(2-chloroethyl)-4-oxoimidazo[5,1-d]-1,2,3,5-tetrazine-8-ca rboxylate but not with nitrogen mustard; (iii) ellagic acid has no effect upon the frequency of alkylation observed with the monofunctional alkylators N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and methylmethanesulfonate; (iv) ellagic acid increases the frequency of depurination and strand break formation induced by CNU without affecting the sequence-selective pattern of depurination.
Topics: Alkylation; Base Sequence; Cross-Linking Reagents; DNA; DNA Damage; Ellagic Acid; Ethylnitrosourea; Guanine; Methyl Methanesulfonate; Molecular Sequence Data; Spermidine
PubMed: 1547533
DOI: 10.1093/carcin/13.3.425