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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 -
Annual Review of Biochemistry Jun 2017Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the... (Review)
Review
Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.
Topics: Adenine; Biological Evolution; Bloom Syndrome; Catalysis; DNA Repair; DNA, Catalytic; DNA, Cruciform; DNA, Single-Stranded; Guanine; Humans; Hydrolysis; Inverted Repeat Sequences; Mutation; Polymorphism, Genetic; Werner Syndrome; beta-Globins
PubMed: 28654322
DOI: 10.1146/annurev-biochem-070611-095951 -
The International Journal of... Jun 2018Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat...
Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat shock and oxidative stresses through stimulation of the synthesis of heat shock and antioxidant proteins. This time, we investigated whether polyamines are directly involved in protecting nucleic acids from thermal depurination induced by high temperature. The suppressing efficiencies of depurination of DNA by spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg, were approximately 50%, 60% and 80%, respectively. Mg also protected nucleic acids against depurination but to a lesser degree than polyamines. Longer unusual polyamines were more effective at protecting DNA against depurination compared to standard polyamines. The tRNA depurination suppressing efficiencies of spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg, were approximately 60%, 70% and 80%, respectively. Standard polyamines protected tRNA and ribosomes more effectively than DNA against thermal depurination. Branched polyamines such as mitsubishine and tetrakis(3-aminopropyl)ammonium also protected RNA more effectively than DNA against depurination. These results suggest that the suppressing effect of depurination of nucleic acids (DNA and RNA) depends on the types of polyamines: i.e. to maintain functional conformation of nucleic acids at high temperature, longer and branched polyamines play important roles in protecting nucleic acids from depurination compared to standard polyamines and Mg.
Topics: DNA; Hot Temperature; Humans; Nucleic Acid Conformation; Polyamines; Purines; RNA, Transfer; Ribosomes
PubMed: 29649565
DOI: 10.1016/j.biocel.2018.04.008 -
PloS One 2014Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study,...
Depurination has attracted considerable attention since a long time for it is closely related to the damage and repair of nucleic acids. In the present study, depurination using a pool of 30-nt short DNA pieces with various sequences at diverse pH values was analyzed by High Performance Liquid Chromatography (HPLC). Kinetic analysis results showed that non-enzymatic depurination of oligodeoxynucleotides exhibited typical first-order kinetics, and its temperature dependence obeyed Arrhenius' law very well. Our results also clearly showed that the linear relationship between the logarithms of rate constants and pH values had a salient point around pH 2.5. Interestingly and unexpectedly, depurination depended greatly on the DNA sequences. The depurination of poly (dA) was found to be extremely slow, and thymine rich sequences depurinated faster than other sequences. These results could be explained to some extent by the protonation of nucleotide bases. Moreover, two equations were obtained based on our data for predicting the rate of depurination under various conditions. These results provide basic data for gene mutagenesis and nucleic acids metabolism in acidic gastric juice and some acidic organelles, and may also help to rectify some misconceptions about depurination.
Topics: Base Sequence; Chromatography, High Pressure Liquid; DNA; Hydrogen-Ion Concentration; Kinetics; Nucleic Acids; Oligodeoxyribonucleotides; Protons; Purines; Salts; Temperature; Time Factors
PubMed: 25546310
DOI: 10.1371/journal.pone.0115950 -
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 -
FEBS Open Bio Nov 2017DNA base depurination is one of the most common forms of DNA damage and , and the suppression of depurination is very important for versatile applications of DNA in...
DNA base depurination is one of the most common forms of DNA damage and , and the suppression of depurination is very important for versatile applications of DNA in biotechnology and medicine. In this work, it was shown that the polycations chitosan (Cho) and spermine (Spm) strongly inhibit DNA depurination through the formation of polyion complexes with DNA molecules. The intramolecular electrostatic interaction of positively charged polycations with DNA efficiently suppresses the protonation of purine groups, which is the key step of depurination. Importantly, the optimal pH for Cho's inhibition of depurination is significantly different from that of Spm. Cho is very effective in the inhibition of depurination in highly acidic media (pH: 1.5-3), whereas Spm is found to suppress the chemical reaction near neutral pH, as well as in acidic solutions. This remarkable pH specificity of the two biorelevant polycations is attributed to the difference in the values of the amino groups. The relevance of our results with the biological roles of biogenic polycations is also discussed.
PubMed: 29123979
DOI: 10.1002/2211-5463.12308 -
Chemistry, An Asian Journal Dec 2022Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase...
Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase (depurination) at the sarcin/ricin loop of the ribosomal RNA. In this work, we describe the preparation and optimization process of a fluorescent probe that can be used to visualize depurination. Starting from a fluorescent thiophene nucleobase analog, various RNA probes that fluoresce exclusively in the presence of a depurinated sarcin/ricin-loop RNA were designed and characterized. The main challenge in this process was to obtain a high fluorescence signal in the hybridized state with an abasic RNA strand, while keeping the background fluorescence low. With our new RNA probes, the fluorescence intensity and lifetime can be used for efficient monitoring of depurinated RNA.
Topics: Ricin; RNA Probes; RNA; Fluorescence; Purines
PubMed: 36321802
DOI: 10.1002/asia.202201077 -
The Analyst Oct 2019Depurination occurs via hydrolysis of the purine-deoxyribose glycosyl bond and causes nucleic acid damage. In particular, the DNA sequences that can undergo a...
Depurination occurs via hydrolysis of the purine-deoxyribose glycosyl bond and causes nucleic acid damage. In particular, the DNA sequences that can undergo a self-catalyzed depurination (SCD) will cause a great uncertainty in duplicating, separating, purifying, and storing the DNA samples. Therefore, there is a great demand to develop a rapid detection method for SCD events. Herein, the use of a convenient fluorescence method to follow the site-specific SCD was demonstrated. We found that the resultant apurine site (AP site) from depurination can be selectively recognized by a fluorescent probe of palmatine (PAL) with a turn-on fluorescence response. The dependence of SCD on the bases of the depurination site, pH, metal ions, and time shows that our method can be used to rapidly evaluate the depurination process. Furthermore, the depurination process can be photo-switched using a photoacid as an external initiator. Our work will find wide applications in preliminarily identifying the DNA depurination.
Topics: Berberine Alkaloids; Catalysis; DNA Glycosylases; DNA, Catalytic; Fluorescent Dyes; Molecular Structure
PubMed: 31482933
DOI: 10.1039/c9an01412h -
Nucleic Acids Research Dec 2008Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that...
Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.
Topics: Bromovirus; Hordeum; Protoplasts; Purines; RNA, Viral; RNA-Dependent RNA Polymerase; Ribosome Inactivating Proteins, Type 1; Virus Replication
PubMed: 19004869
DOI: 10.1093/nar/gkn896 -
Nucleic Acids Research Sep 2011Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain...
Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.
Topics: Bromovirus; Capsid Proteins; Protoplasts; Purines; RNA, Viral; Ribosome Inactivating Proteins, Type 1; Temperature; Virion; Virus Assembly
PubMed: 21609957
DOI: 10.1093/nar/gkr383