title: Detection of oxidation products of 5-methyl-2'-deoxycytidine in Arabidopsis DNA.
abstract: Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2'-deoxycytidine (5-FodC), 5-carboxyl-2'-deoxycytidine (5-CadC), 5-hydroxymethyl-2'-deoxyuridine (5-HmdU), and the (5'S) diastereomer of 8,5'-cyclo-2'-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 10(6) nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis.
authors.affiliation: Environmental Toxicology Graduate Program, University of California Riverside, Riverside, California, United States of America.
authors.forename: Shuo
authors.initials: S
authors.lastname: Liu
authors.affiliation: Beckman Research Institute, City of Hope Medical Centre, Duarte, California, United States of America.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Dunwell
authors.affiliation: Beckman Research Institute, City of Hope Medical Centre, Duarte, California, United States of America.
authors.forename: Gerd P
authors.initials: GP
authors.lastname: Pfeifer
authors.affiliation: School of Agriculture, Policy and Development, University of Reading, Reading, Berkshire, United Kingdom.
authors.forename: Jim M
authors.initials: JM
authors.lastname: Dunwell
authors.affiliation: School of Agriculture, Policy and Development, University of Reading, Reading, Berkshire, United Kingdom ; Agricultural Biotechnology Research Institute, Faisalabad, Pakistan.
authors.forename: Ihsan
authors.initials: I
authors.lastname: Ullah
authors.affiliation: Environmental Toxicology Graduate Program, University of California Riverside, Riverside, California, United States of America ; Department of Chemistry, University of California Riverside, Riverside, California, United States of America.
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D018744:DNA, Plant / Q000737:chemistry*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003843:Deoxycytidine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000235:genetics / Q000502:physiology*; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D015394:Molecular Structure; D009705:Nucleosides / Q000378:metabolism*; D010084:Oxidation-Reduction; D053719:Tandem Mass Spectrometry; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: C000600732:5-carboxyl-2'-deoxycytidine; C402104:5-formyl-2'-deoxycytidine; D018744:DNA, Plant; D005452:Fluoresceins; D009705:Nucleosides; D003841:Deoxycytidine; D003843:Deoxycytidine Monophosphate; C079259:5-hydroxymethyldeoxycytidylic acid; C018080:5-hydroxymethyl-2'-deoxyuridine; C084577:5-carboxyfluorescein diacetate; C016569:5-methyldeoxycytidine; D013936:Thymidine