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The Journal of Biological Chemistry Oct 2011The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The...
The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The variation in self-depurination kinetics with sequence changes in the loop residues and stem base pairs, as well as with pH, provides insights into the self-catalytic mechanism. The observations suggest that self-catalyzed depurination of the 5' G residue of the loop consensus sequence 5'-G(T/A)GG-3' probably involves formation of some intraloop hydrogen-bonded base pair with the 3'-terminal G residue; although the electronic structure of both these G residues is retained, their 2-amino substituents are not critical for that interaction. The strong dependence of the self-depurination kinetics on stem stability suggests that the lifetime of some strained form of the loop is controlled by the integrity of the stem. In addition to the effects of length and base pair sequence on stem stability, there is a base pair requirement at the base of the loop: self-depurination is suppressed by 5'-C·G-3', 5'-A·T-3', or a mismatch but is most favored by 5'T·A3' and less so by 5'-G·C-3'. The occurrence in T and G of a similarly located carbonyl capable of hydrogen-bonding to the water molecule required for glycosyl bond hydrolysis may explain this sequence requirement. In toto, the more complete definition of the consensus sequence provided by this investigation enables a more accurate estimation of their number in the human genome and their distribution among different genes.
Topics: Catalysis; DNA; Genome, Human; Humans; Hydrogen Bonding; Hydrogen-Ion Concentration; Kinetics; Nucleic Acid Conformation; Purines
PubMed: 21868376
DOI: 10.1074/jbc.M111.272047 -
Nucleic Acids Research Sep 1994DNA was alkylated with nitrogen mustard (HN2) and the rate of release of the alkylpurines was quantitated by HPLC. The half life of depurination of the major product...
DNA was alkylated with nitrogen mustard (HN2) and the rate of release of the alkylpurines was quantitated by HPLC. The half life of depurination of the major product (7-alkylguanine) was 9.1 h at 37 degrees C. End-labelled DNA was used to show that depurination occurred dominantly at 5'-GA, 5'-GG and 5'-GT sequences. Although extensive alkylation was observed at all 5'-GNC and 5'GNT sequences, no depurination was observed at these sites during a depurination time of 20 h at 37 degrees C. Since these sites are potential interstrand crosslinking sequences (G-adduct-G and G-adduct-A, both spanning an intervening base pair), this suggests that these regions have a greatly enhanced stability or that simultaneous depurination of both ends of the crosslink is necessary before these lesions are removed (with a predicted half-life of approximately 80 h at 37 degrees C). Depurination at the lac UV5 promoter impaired the association of Escherichia coli RNA polymerase with that promoter, while in the elongation phase two distinctly different sequence-specific processes were apparent. At 5'-GNC and 5'-GNT sequences transcriptional blockages were maintained with increasing elongation time, whereas at monoadduct sites, the blockage decreased with elongation time (predominantly at 5'-GG and 5'-GC sequences), with an average half-life of approximately 10.7 h. Collectively, these results suggest that the observed read-through past monoadduct sites is due to depurination of the DNA at those sites. E. coli RNA polymerase is therefore able to transcribe efficiently past apurinic sites and presumably does so by incorporating an incorrect base into the nascent RNA.
Topics: Alkylation; Animals; Base Sequence; Cattle; DNA; DNA-Directed RNA Polymerases; Escherichia coli; Guanine; Half-Life; Kinetics; Mechlorethamine; Molecular Sequence Data; Promoter Regions, Genetic; Purines; RNA, Messenger; Transcription, Genetic
PubMed: 7937107
DOI: 10.1093/nar/22.19.3880 -
Proceedings of the National Academy of... Mar 2006A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The...
A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The critical features of that catalytic intermediate are a 5'-G-T-G-G-3' loop and an adjacent 5'-T.A-3' base pair of a short duplex stem stable enough to fix the loop structure required for depurination of its 5'-G residue. That residue is uniquely depurinated with a rate some 5 orders of magnitude faster than that of random "spontaneous" depurination. In contrast, all other purine residues in the sequence depurinate at the spontaneous background rate. The reaction requires no divalent cations or other cofactors and occurs under essentially physiological conditions. Such stem-loops can form in duplex DNA under superhelical stress, and their critical sequence features have been found at numerous sites in the human genome. Self-catalyzed stem-loop-mediated depurination leading to flexible apurinic sites may therefore serve some important biological role, e.g., in nucleosome positioning, genetic recombination, or chromosome superfolding.
Topics: Base Sequence; Catalysis; DNA; Genes; Genome, Human; Guanine; Humans; Nucleic Acid Conformation; Single-Strand Specific DNA and RNA Endonucleases
PubMed: 16537362
DOI: 10.1073/pnas.0508499103 -
Toxins Mar 2018We wish to make the following correction to the published paper [1].[...].
We wish to make the following correction to the published paper [1].[...].
PubMed: 29494495
DOI: 10.3390/toxins10030107 -
Nucleic Acids Research May 2010We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The...
We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies' SurePrint DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology).
Topics: Indicators and Reagents; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Oligonucleotides; Polymerase Chain Reaction; Purines
PubMed: 20308161
DOI: 10.1093/nar/gkq163 -
The International Journal of... Dec 2012The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces...
A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition, cell signaling and apoptosis in mammalian cells.
The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.
Topics: Animals; Apoptosis; Caspase 3; Caspase 7; Cattle; Cell Line; Enzyme Activation; Humans; JNK Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Mutation, Missense; Nucleosomes; Protein Biosynthesis; Protein Sorting Signals; Ribosomes; Ricin; Signal Transduction; p38 Mitogen-Activated Protein Kinases
PubMed: 22982239
DOI: 10.1016/j.biocel.2012.09.004 -
Mini Reviews in Medicinal Chemistry Jun 2004Pokeweed antiviral protein and several other ribosome inactivating proteins are effective against a broad range of viruses. Recent results have shown that their... (Review)
Review
Pokeweed antiviral protein and several other ribosome inactivating proteins are effective against a broad range of viruses. Recent results have shown that their enzymatic activity is not limited to depurination of the large rRNA, they can depurinate other nucleic acids, including viral RNAs. Antiviral activity of RIPs is summarized here in light of their novel activities and recent developments in the field.
Topics: Animals; Antiviral Agents; Humans; Nucleic Acids; Proteins; Ribosomes; Viruses
PubMed: 15180509
DOI: 10.2174/1389557043403800 -
The Journal of Biological Chemistry Apr 2013The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze...
The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze depurination of the 5'G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease.
Topics: Anemia; Animals; Codon; DNA; Humans; Mutation; Nucleic Acid Conformation; Thalassemia; beta-Globins
PubMed: 23457306
DOI: 10.1074/jbc.M113.454744 -
Molecular Pharmacology Nov 1966
Topics: Adenine; Alkylation; Animals; Cattle; Chromatography, Paper; DNA; DNA, Bacterial; Escherichia coli; Guanine; Hot Temperature; Hydrogen-Ion Concentration; Mutation; Purines; RNA; Sarcina; Thymus Gland
PubMed: 5337732
DOI: No ID Found -
Journal of Biotechnology Jan 2009Ricin A-chain can inactivate eukaryotic ribosomes, but exhibits no N-glycosidase activity on intact E. coli ribosomes. In the present research, in order to avoid using... (Comparative Study)
Comparative Study
Comparative analysis of depurination catalyzed by ricin A-chain on synthetic 32mer and 25mer oligoribonucleotides mimicking the sarcin/ricin domain of the rat 28S rRNA and E. coli 23S rRNA.
Ricin A-chain can inactivate eukaryotic ribosomes, but exhibits no N-glycosidase activity on intact E. coli ribosomes. In the present research, in order to avoid using radiolabeled oligoribonucleotides, two kinds of synthetic 5'-FAM fluorescence-labeled oligoribonucleotide substrates were used to mimic the sarcin/ricin domains of rat 28S rRNA and E. coli 23S rRNA (32mer and 25mer, named as Rat FAM-SRD and E. coli FAM-SRD, respectively). Ricin A-chain was able to specifically release adenine from the first adenosine of the GAGA tetraloop and exhibited specific N-glycosidase activity under neutral and weak acidic conditions with both substrates. However, under more acidic conditions, ricin A-chain was able to release purines from other sites on eukaryotic substrates, but it retained specific depurination activity on prokaryotic substrates. At pH 5.0, the Michaelis constant (K(m)) for the reaction with Rat FAM-SRD (4.57+/-0.28microM) corresponded to that with E. coli FAM-SRD (4.64+/-0.26microM). However, the maximum velocity (V(max)) for ricin A-chain with Rat FAM-SRD was 0.5+/-0.024microM/min, which is higher than that with E. coli FAM-SRD (0.32+/-0.011microM/min).
Topics: Animals; Escherichia coli; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Nucleic Acid Conformation; Oligoribonucleotides; Purines; RNA, Bacterial; RNA, Ribosomal, 23S; RNA, Ribosomal, 28S; Rats; Ricin
PubMed: 19014981
DOI: 10.1016/j.jbiotec.2008.10.005