{"doi":"10.1016/j.jbc.2022.101638","title":"Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase","abstract":"The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts. The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts. Cytosine to thymine transition mutations are the most abundant single-base changes observed in human cancer cells (1Hollstein M. Sidransky D. Vogelstein B. Curtis H.C. p53 mutations in human cancers.Science. 1991; 253: 49-53Google Scholar, 2Magewu A.N. Jones P.A. Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.Mol. Cell. Biol. 1994; 14: 4225-4232Google Scholar, 3Iengar P. An analysis of substitution, deletion and insertion mutations in cancer genes.Nucleic Acids Res. 2012; 40: 6401-6413Google Scholar, 4Forbes S.A. Beare D. Boutselakis H. Bamford S. Bindal N. Tate J. Cole C.G. Ward S. Dawson E. Ponting L. Stefancsik R. Harsha B. YinKok C. Jia M. Jubb H. et al.Cosmic: Somatic cancer genetics at high-resolution.Nucleic Acids Res. 2017; 45: D777-D783Google Scholar, 5Lewis C.A. Crayle J. Zhou S. Swanstrom R. Wolfenden R. Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 8194-8199Google Scholar). These mutations are believed to arise from the hydrolytic deamination of cytosine and cytosine analogs, which involves a water molecule adding to the C4 carbon, displacing the amino group (6Lindahl T. Nyberg B. Heat-induced deamination of cytosine residues in DNA.Biochemistry. 1974; 13: 3405-3410Google Scholar, 7Coulondre C. Miller J.H. Farabaugh P.J. Gilbert W. Molecular basis of ","journal":"Journal of Biological Chemistry","year":2022,"id":288809,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":6,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9551,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":872355,"name":"Mark L. 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Sowers","orcid":"0000-0002-8402-4792","position":7,"is_corresponding":false},{"id":913263,"name":"Chia Wei Hsu","orcid":"0000-0001-8863-462X","position":0,"is_corresponding":true}],"reference_count":72,"raw_metadata":null,"created_at":"2026-07-19T00:30:14.968626Z","pmid":"35085553","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}