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The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ[subscript 0]) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2’-deoxy-preQ[subscript 0] and 2’-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ[subscript 0] derivative, 2’-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism., Deutsche Forschungsgemeinschaft, Singapore-MIT Alliance in Research and Technology (SMART)

Although mechanistically linked to disease, cellular molecules damaged by endogenous processes have not emerged as significant biomarkers of inflammation and disease risk, due in part to poor understanding of their pharmacokinetic fate from tissue to excretion. Here, we use systematic metabolite profiling to define the fate of a common DNA oxidation product, base propenals, to discover such a biomarker. Based on known chemical reactivity and metabolism in liver cell extracts, 15 candidate metabolites were identified for liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) quantification in urine and bile of rats treated with thymine propenal (Tp). Analysis of urine revealed three metabolites (6% of Tp dose): thymine propenoate and two mercapturate derivatives of glutathione conjugates. Bile contained an additional four metabolites (22% of Tp dose): cysteinylglycine and cysteine derivatives of glutathione adducts. A bis-mercapturate was observed in urine of untreated rats and increased approximately three- to fourfold following CCl[subscript 4]-induced oxidative stress or treatment with the DNA-cleaving antitumor agent, bleomycin. Systematic metabolite profiling thus provides evidence for a metabolized DNA damage product as a candidate biomarker of inflammation and oxidative stress in humans., National Cancer Institute (U.S.) (CA026731)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014., This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections., Cataloged from student-submitted PDF version of thesis., Includes bibliographical references., The work presented in this thesis explores two novel and complementary facets of endogenous DNA damage: the development of biomarkers of inflammation based on metabolites of DNA damage products and the formation of DNA adducts by electrophilic products of intermediary metabolism. From the first perspective, endogenous DNA damage generated by reactive oxygen and nitrogen species from inflammation and oxidative stress has shown strong mechanistic links to the pathophysiology of cancer and other human diseases, with the damage products reflecting all types of damage chemistries including oxidation, deamination, halogenation, nitration and alkylation. However, the use of DNA damage products as biomarkers has been limited by poor understanding of the damage actually arising in tissues and a lack of appreciation of the fate of DNA damage products from the moment of formation at the site of damage to release from cells to final excretion from the body. The goal of the work presented in the first part of this thesis was to investigate the metabolic fates of the base propenal products arising from 4'-oxidation of 2'-deoxyribose in DNA, one of the most common products of DNA oxidation, and to define base propenal metabolites as potential biomarkers of oxidative stress. This project was approached with systematic metabolite profiling, starting with prediction of potential base propenal metabolites based on a priori knowledge of its chemical reactivity as an [alpha],[beta]-unsaturated aldehyde toward glutathione (GSH) in non-enzymatic reactions and in rat liver cell extracts. Of 15 potential candidates predicted and identified from these in vitro studies, analysis of urine samples from rats given intravenous doses (IV) of thymine propenal revealed three major metabolites: thymine propenoic acid and two mercapturic acid derivatives, which accounted for ~6% of the injected dose. An additional four metabolites, including conjugates with GSH, cysteinylglycine and cysteine, were obs, by Watthanachai Jumpathong., Ph. D.