Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

• Published in: The Journal of biological chemistry 2014-07, Vol.289 (29), p.20273-20282
• Main Authors: Bullard, Whitney, Lopes da Rosa-Spiegler, Jessica, Liu, Shuo, Wang, Yinsheng, Sabatini, Robert
• Format: Article
• Language: English
• Subjects: Base J; DNA and Chromosomes; DNA Enzyme; DNA, Protozoan - chemistry; DNA, Protozoan - genetics; DNA, Protozoan - metabolism; Epigenesis, Genetic; Epigenetics; Gene Knockout Techniques; Genome, Protozoan; Glucosides - chemistry; Glucosides - metabolism; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Glycosyltransferase; Mutagenesis, Site-Directed; Parasite; Pentoxyl - analogs & derivatives; Pentoxyl - chemistry; Pentoxyl - metabolism; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; RNA Interference; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Protozoan - genetics; RNA, Protozoan - metabolism; Substrate Specificity; Thymine - chemistry; Thymine - metabolism; Transcription Regulation; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology; Trypanosoma brucei brucei - genetics; Trypanosomatid; Uracil - analogs & derivatives; Uracil - chemistry; Uracil - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.579821

O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. β-d-glucopyranosyloxymethyluracil (base J) synthesis is initiated by the JBP1/2 enzymes that hydroxylate thymine, forming 5-hydroxymethyluracil (hmU). hmU is then glucosylated by a previously unknown glucosyltransferase. A recent computational screen identified a possible candidate for the base J-associated glucosyltransferase (JGT) in trypanosomatid genomes. We demonstrate that recombinant JGT utilizes uridine diphosphoglucose to transfer glucose to hmU in the context of dsDNA. Mutation of conserved residues typically involved in glucosyltransferase catalysis impairs DNA glucosylation in vitro. The deletion of both alleles of JGT from the genome of Trypanosoma brucei generates a cell line that completely lacks base J. Reintroduction of JGT in the JGT KO restores J synthesis. Ablation of JGT mRNA levels by RNAi leads to the sequential reduction in base J and increased levels of hmU that dissipate rapidly. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only glucosyltransferase enzyme required for the second step of the pathway. Similar to the activity of the related Ten-Eleven Translocation (TET) family of dioxygenases on 5mC, our studies also suggest the ability of the base J-binding protein enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. Here we discuss the regulation of hmU and base J formation in the trypanosome genome by JGT and base J-binding protein. Background: Base J regulates Pol II transcription in trypanosomatids and is synthesized in two steps involving DNA hydroxylation and glucosylation. Results: JGT catalyzes the transfer of glucose from uridine diphosphoglucose to hmU in DNA. Conclusion: JGT is the glucosyltransferase involved in base J synthesis. Significance: JGT is the first known glucosyltransferase that modifies eukaryotic DNA.