Regulation of DNA glycosylation by two thymidine hydroxylase enzymes in Trypanosoma brucei
Birkeland, Shanda Raye
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Base J, or [beta]-D-glucosyl-hydroxymethyluracil, is an unusual DNA base modification present in the genome of bloodstream form Trypanosoma brucei. The presence of J correlates with epigenetic silencing of telomeric expression sites regulating antigenic variation. Two proteins, JBP1 and JBP2, are hypothesized to regulate the first step in J biosynthesis via a conserved N-terminal AlkB-like thymidine hydroxylase (TH) domain. However, the functional domains and binding requirements for JBP1 and JBP2 stimulated hydroxylation remain to be tested. Mutagenesis, domain swapping, and heterologous expression experiments suggest a role of DNA substrate specificity for thymidine hydroxylation. These data show the JBP2 TH motif is essential for function and JBP1 and JBP2 are TH enzymes with non-interchangeable domains. Furthermore, JBP2 homologs show decreased levels of J synthesis in different DNA substrates. These results highlight fundamental differences in thymidine hydroxylation by JBP1 and JBP2 in J biosynthesis.