Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome [Glycobiology and Extracellular Matrices]

June 2nd, 2014 by Bullard, W., Lopes da Rosa-Spiegler, J., Liu, S., Wang, Y., Sabatini, R.

O-linked glucosylation of thymine in DNA (base J) is an important regulatory epigenetic mark in trypanosomatids. 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 GT (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 GT 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 rapidly dissipates. The analysis of JGT function confirms the two-step J synthesis model and demonstrates that JGT is the only GT enzyme required for the second step of the pathway. Similar to the activity of the related TET family of dioxygenases on 5mC, our studies also suggest the ability of the JBP enzymes to catalyze iterative oxidation of thymine in trypanosome DNA. The regulation of hmU and base J formation in the trypanosome genome by JGT and JBP will be discussed.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Identification of the glucosyltransferase that converts hydroxymethyluracil to base J in the trypanosomatid genome [Glycobiology and Extracellular Matrices]