title: Quantitative mass spectrometry-based analysis of β-D-glucosyl-5-hydroxymethyluracil in genomic DNA of Trypanosoma brucei.
abstract: β-D-glucosyl-5-hydroxymethyluracil (base J) is a hyper-modified nucleobase found in the nuclear DNA of kinetoplastid parasites. With replacement of a fraction of thymine in DNA, J is localized primarily in telomeric regions of all organisms carrying this modified base. The biosynthesis of J occurs in two putative steps: first, a specific thymine in DNA is recognized and converted into 5-hydroxymethyluracil (5-HmU) by J-binding proteins (JBP1 and JBP2); a glucosyl transferase (GT) subsequently glucosylates the 5-HmU to yield J. Although several recent studies revealed the roles of internal J in regulating transcription in kinetoplastids, functions of telomeric J and proteins involved in J synthesis remain elusive. Assessing the functions of base J and understanding fully its biosynthesis necessitate the measurement of its level in cells and organisms. In this study, we reported a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS) method, together with the use of a surrogate internal standard (β-D-glucosyl-5-hydroxymethyl-2'-deoxycytidine, 5-gHmdC), for the accurate detection of β-D-glucosyl-5-hydroxymethyl-2'-deoxyuridine (dJ) in Trypanosoma brucei DNA. For comparison, we also measured the level of the precursor for dJ synthesis [i.e. 5-hydroxymethyl-2'-deoxyuridine (5-HmdU)]. We found that base J was not detectable in the JBP-null cells whereas it replaced approximately 0.5% thymine in wild-type cells, which was accompanied with a markedly decreased level of 5-HmdU in JBP1/JBP2-null strain relative to the wild-type strain. These results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ. This is the first report about the application of LC-MS/MS for the quantification of base J. The analytical method built a solid foundation for dissecting the molecular mechanisms of J biosynthesis and assessing the biological functions of base J in the future.ᅟ
authors: Environmental Toxicology Graduate Program, University of California, Riverside, CA, 92521, USA.
authors: Shuo
authors: S
authors: Liu
authors: Debin
authors: D
authors: Ji
authors: Laura
authors: L
authors: Cliffe
authors: Robert
authors: R
authors: Sabatini
authors: Yinsheng
authors: Y
authors: Wang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D016054:DNA, Protozoan / Q000737:chemistry*; D005960:Glucosides / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014346:Trypanosoma brucei brucei / Q000737:chemistry*; D014498:Uracil / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry
chemical_list: D016054:DNA, Protozoan; D005960:Glucosides; C084880:5-((glucopyranosyloxy)methyl)uracil; D014498:Uracil