We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by proteins (JBP1 and JBP2) homologous to the Fe(2+)/2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxylation is driven by the oxidative decarboxylation of 2-OG, forming succinate and CO(2). However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demonstrate recombinant JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe(2+)-, 2-OG-, and O(2)-dependent manner. Under anaerobic conditions, ... More
We have recently demonstrated that O-linked glucosylation of thymine in trypanosome DNA (base J) regulates polymerase II transcription initiation. In vivo analysis has indicated that base J synthesis is initiated by the hydroxylation of thymidine by proteins (JBP1 and JBP2) homologous to the Fe(2+)/2-oxoglutarate (2-OG)-dependent dioxygenase superfamily where hydroxylation is driven by the oxidative decarboxylation of 2-OG, forming succinate and CO(2). However, no direct evidence for hydroxylase activity has been reported for the JBP proteins. We now demonstrate recombinant JBP1 hydroxylates thymine specifically in the context of dsDNA in a Fe(2+)-, 2-OG-, and O(2)-dependent manner. Under anaerobic conditions, the addition of Fe(2+) to JBP1/2-OG results in the formation of a broad absorption spectrum centered at 530 nm attributed to metal chelation of 2-OG bound to JBP, a spectroscopic signature of Fe(2+)/2-OG-dependent dioxygenases. The N-terminal thymidine hydroxylase domain of JBP1 is sufficient for full activity and mutation of residues involved in coordinating Fe(2+) inhibit iron binding and thymidine hydroxylation. Hydroxylation in vitro and J synthesis in vivo is inhibited by known inhibitors of Fe(2+)/2-OG-dependent dioxygenases. The data clearly demonstrate the JBP enzymes are dioxygenases acting directly on dsDNA, confirming the two-step J synthesis model. Growth of trypanosomes in hypoxic conditions decreases JBP1 and -2 activity, resulting in reduced levels of J and changes in parasite virulence previously characterized in the JBP KO. The influence of environment upon J biosynthesis via oxygen-sensitive regulation of JBP1/2 has exciting implications for the regulation of gene expression and parasite adaptation to different host niches.
