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 while 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.

Insights into the Activity Change of Spore Photoproduct Lyase Induced by Mutations at a Peripheral Glycine Residue

“…As revealed in previous studies (Ciccimaro and Blair, 2010; Liu et al, 2014; Wagner et al, 2015), linear responses between the intensity of mass spec signals and the amount of the analytes were observed, demonstrating the feasibility of using this LC-MRM-MS assay for quantitative analysis. The HPLC experiments used a ZORBAX Eclipse plus C18 column 4.6 × 50 mm (3.5 μm in particle size, Agilent Technologies, Santa Clara, CA) at ambient temperature, following the experimental procedures described in our previous studies (Yang et al, 2017).…”

Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing

“…SMRT sequencing was indispensable for identifying the exact locations of J in DNA. Immunoprecipitation of J-containing DNA fragments lacks resolution and the recently developed liquid chromatography–mass spectrometry/mass spectrometry method to detect J ( 6 ) is also unsuitable for localization. Attempts to develop (bio)chemical procedures targeting J have failed thus far.…”

“…Base J has been found in all kinetoplastid flagellates analyzed ( 4 ), including major pathogens such as Trypanosoma brucei, Trypanosma cruzi and Leishmania species, and in the related unicellular alga, Euglena ( 5 ). In all these organisms, base J replaces 0.5–1% of T, mainly in telomeric repeats ( 4 , 6 ) and other repetitive sequences ( 7 ).…”

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

“…Along this line, Wang and co-workers 199 recently developed an assay for the quantification of β- d -glucosyl-5-hydroxymethyl-2′-deoxyuridine (dJ) in Trypanosoma brucei DNA, where a surrogate standard, p-D-glucosyl-5-hydroxymethyl-2′-deoxycytidine, was employed to avoid pursuing a multi-step synthesis of an isotopically labeled dJ. From the data acquired on three separate days, excellent linearity ( R 2 = 0.998) was observed for the calibration curve.…”