Quantifizierung und Charakterisierung der DNA-Phosphorothioat Modifikation in der Darmflora
Zusammenfassung der Projektergebnisse
There are currently >120 different chemical modifications of the canonical nucleosides in RNA and DNA in all forms of life. In both prokaryotes and eukaryotes, DNA modifications represent a second layer of genetic information as epigenetic marks to control gene expression, while bacterial genomes are also uniquely modified in restriction-modification systems. Modified ribonucleosides also occur in all known forms of RNA, representing 10% of the nucleotide content of tRNA molecules. For example, 7-deazaguanosine modifications, such as queuosine, are present in tRNA in both prokaryotes and eukaryotes. As a demonstration of the surprising plasticity of nucleic acid modifications, we recently used a combination of comparative genomics and chromatography-coupled mass spectrometry (LC-MS) to discover queuosine-like DNA modifications in bacteria. Genomic analysis revealed paralogs of tRNA guanosine transglycosylase genes (tgtA5) alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, which led to the hypothesis that 7-deazaguanine derivatives are inserted into DNA. This prediction was tested using neutral loss scanning LC-MS to discover 2’-deoxy-7-amido-7-deazaguanosine (dADG), and its putative precursor 2’-deoxy-preQ0, in enzymatic hydrolysates of DNA extracted from tgtA5-containing Salmonella enterica serovar Montevideo. Subsequent high mass-accuracy MS was used to verify the structures of dpreQ0 and dADG. Quantitation using synthetic standards revealed 1 dADG per 1000 nucleotides. The tgtA5 gene cluster has now been found in >150 phylogenetically diverse bacteria, with evidence for its participation in restriction-modification (RM) systems in some cases. Another example of the diversity of bacterial DNA modifications involves phosphorothioation (PT) of DNA, in which sulfur replaces a non-bonding oxygen in the DNA backbone. PTs have been found in >300 bacterial species and are inserted by a horizontally-transferred, five-gene dnd cluster (dndA-E). Like dADG, PT appears to have both: RM functions coupled with dndF-H restriction genes, and non-RM functions in bacteria lacking those restriction genes. To understand better PT biochemistry in vivo, we combined in vivo isotopic labeling with sensitive MS methods to define the chemical behavior of PTs during oxidative stress and other conditions of cell growth. We did not observe a resistance of PT-containing bacteria during exposure with the oxidants H2O2, in contrast to predictions from published studies. However, we found that PT conferred significant sensitivity to strand breaks caused by PT oxidation due to the neutrophil-derived chemical mediator of inflammation HOCl. Using a sensitive LC-MS/MS method for the identification and quantification of all 16 possible PT dinucleotides in a limit nuclease digest of DNA, we made the surprising finding of PT modifications in at least 10% of the bacterial genomes isolated from the murine gut microbiome. Interestingly, we did not find dADG or dpreQ0 modifications. The apparent sensitivity of PT-containing bacteria to chemical mediators of inflammation led us to assess changes in the PT levels, occurring during dextran sodium sulfate-induced colitis, in mice. We observed an increase in the level of many PT dinucleotides in direct correlation with inflammation pathology and microbiome changes, which suggests a possible selective advantage for PT-containing bacteria in their natural environment, in spite of the stressful conditions of inflammation. Given the bacterial specificity of the PT and dADG R-M modification systems and their widespread presence in bacterial pathogens, the enzymes synthesizing these DNA modifications represent novel targets for antibiotics.
Projektbezogene Publikationen (Auswahl)
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Novel genomic island modifies DNA with 7-deazaguanine derivatives. Proc Natl Acad Sci USA. 2016 Feb 29
Thiaville JJ, Kellner SM, Yuan Y, Hutinet G, Thiaville PC, Jumpathong W, Mohapatra S, Brochier-Armanet C, Letarov AV, Hillebrand R, Malik CK, Rizzo CJ, Dedon PC, de Crécy-Lagard V
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Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability. Nature Chemical Biology volume 13, pages 888–894 (2017)
Stefanie Kellner, Michael S. DeMott, Ching Pin Cheng, Brandon Russell, Bo Cao, Delin You & Peter C. Dedon
