O‐Glycoside Orientation Is an Essential Aspect of Base J Recognition by the Kinetoplastid DNA‐Binding Protein JBP1

Grover, Rajesh K.; Pond, Stephanie J. K.; Cui, Qizhi; Subramaniam, Prem S.; Case, David A.; Millar, David P.; Wentworth, Paul

doi:10.1002/anie.200604635

Cited by 24 publications

(36 citation statements)

References 28 publications

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“…Although synthetic methods were previously developed for the synthesis of dJ and for its incorporation into ODNs [32-34], the methods involve a multi-step synthesis and are not readily amenable for the preparation of stable isotope-labeled dJ. Thus, we decided to take an alternative approach for the LC-MS/MS quantification of dJ in cellular DNA, where we employed a 5-gHmdC-bearing single-stranded ODN as a surrogate internal standard.…”

Section: Resultsmentioning

confidence: 99%

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Liu

Cliffe

et al. 2014

J. Am. Soc. Mass Spectrom.

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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.

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Section: Resultsmentioning

confidence: 99%

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Liu

Cliffe

et al. 2014

J. Am. Soc. Mass Spectrom.

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“…We propose that it recognizes specific regions of chromatin where it can then open up the chromatin via the C-terminal SWI2/SNF2 domain, allowing access of the TH domain to thymidine residues in double stranded DNA. JBP1, on the other hand, is able to bind J-DNA with high affinity (7,22). Once bound to regions of the genome containing base J, JBP1 can hydroxylate adjacent thymidine residues.…”

Section: Discussionmentioning

confidence: 99%

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes

Cliffe

Kieft

Southern

et al. 2009

Nucleic Acids Research

106

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Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (β-d-glucosyl-HOMedU). This modified base is localized primarily to repetitive DNA, namely the telomeres, and is implicated in the regulation of antigenic variation. The base is synthesized in a two-step pathway. Initially, a thymidine residue in DNA is hydroxylated by a thymidine hydroxylase (TH). This intermediate (HOMedU) is then glucosylated to form base J. Two proteins involved in J synthesis, JBP1 (J binding protein 1) and JBP2, contain a putative TH domain related to the family of Fe2+/2-oxoglutarate-dependent hydroxylases. We have previously shown that mutations in the TH domain of JBP1 kill its ability to stimulate J synthesis. Here we show that mutation of key residues in the TH domain of JBP2 ablate its ability to induce de novo J synthesis. While the individual JBP1 null and JBP2 null trypanosomes have reduced J levels, the deletion of both JBP1 and JBP2 generates a cell line that completely lacks base J but still contains glucosyl-transferase activity. Reintroduction of JBP2 in the J-null trypanosome stimulates HOMedU formation and site-specific synthesis of base J. We conclude that JBP2 and JBP1 are the TH enzymes involved in J biosynthesis.

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“…Diethyl ((methylsulfonyl)methyl)phosphonate 6 was prepared according to known procedures [7], while Horner reagent 9 was obtained by reacting half an equivalent of chlorodiethylphosphate with bis-lithiated 8 [8,9]. 3'-Silylated thymidine 12 was produced by a selective cleavage of the primary TBDMS group on 11 [10,11].…”

Section: Chemistrymentioning

confidence: 99%

“…The filtrate was concentrated and the residue purified by flash column chromatography (8)(9)(10)(11)(12) % MeOH + 0.5% HCOOH in CH 2 Cl 2 ) to afford 47 as a white solid (45 mg, 87%). 1 H NMR…”

Section: -((2rmentioning

confidence: 99%

Synthesis and evaluation of 5′-modified thymidines and 5-hydroxymethyl-2′-deoxyuridines as Mycobacterium tuberculosis thymidylate kinase inhibitors

Toti¹,

Verbeke²,

Risseeuw³

et al. 2013

Bioorganic & Medicinal Chemistry

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We report the synthesis of 5'-modified thymidines (16, 18, 21, 23) and 5,5'-bis-substituted 2'-deoxyuridine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 5'-modified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of protecting groups for the 5-CH 2 OH moiety of 2'-deoxyuridines is described that enables to introduce the desired 5'-modification.

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O‐Glycoside Orientation Is an Essential Aspect of Base J Recognition by the Kinetoplastid DNA‐Binding Protein JBP1

Cited by 24 publications

References 28 publications

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

Quantitative Mass Spectrometry-Based Analysis of β-D-Glucosyl-5-Hydroxymethyluracil in Genomic DNA of Trypanosoma brucei

JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes

Synthesis and evaluation of 5′-modified thymidines and 5-hydroxymethyl-2′-deoxyuridines as Mycobacterium tuberculosis thymidylate kinase inhibitors

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