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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 are either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exists 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, hyTDG, which is comprised of a 29-amino acid sequence from human thymine DNA glycosylase linked to a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hyTDG 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 identification and quantification. Using this approach, we have measured for the first time the levels of total Uracil (U), U:G and T:G 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Kangling Zhang

LAB HEAD: Lawrence Sowers

PROVIDER: PXD030459 | Pride | 2022-02-15

REPOSITORIES: Pride

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			Action	DRS
	6XHis_HTDG_82_100_-tTDG_2.mgf	Mgf
	6XHis_HTDG_82_100_-tTDG_2.raw	Raw
	6XHis_HTDG_82_100_-tTDG_4.mgf	Mgf
	6XHis_HTDG_82_100_-tTDG_4.raw	Raw
	6XHis_HTDG_82_100_-tTDG__232.mgf	Mgf

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