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

Abstract

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.

Iron metabolism in trypanosomatids, and its crucial role in infection

“…These mutations do not affect the DNA binding of the enzyme and so the inference is that they affect the catalytic capability, as would be expected if they were required for iron incorporation. Similar mutagenesis studies on iron and 2-oxoglutarate binding residues, also confirm that JBP2 is a member of this family (Cliffe et al 2009 ;Vainio et al 2009). In neither case has the hydroxylase activity been reconstituted in vitro.…”

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

“…90-13 cells were cultured in the presence of 2.5 g/ml neomycin and 5 g/ml hygromycin to maintain the intergrated genes for T7 RNA polymerase and the tetracycline repressor, respectively. Transfections of bloodstream form T. brucei were essentially carried out as described previously (14).…”

“…JBP2 does not bind the modified base directly, but is able to bind chromatin in a base J-independent manner, presumably via the C-terminal SWI2/SNF2 domain (9). Although both JBP1 and JBP2 stimulate de novo thymidine hydroxylation in vivo, the ability of JBP1 to bind J-DNA is thought to play a role in J propagation/maintenance (9,14,19). Deletion of either JBP1 or JBP2 from the bloodstream form T. brucei results in a 20-and 8-fold reduction in the levels of base J, respectively (10,14,20).…”

“…Although both JBP1 and JBP2 stimulate de novo thymidine hydroxylation in vivo, the ability of JBP1 to bind J-DNA is thought to play a role in J propagation/maintenance (9,14,19). Deletion of either JBP1 or JBP2 from the bloodstream form T. brucei results in a 20-and 8-fold reduction in the levels of base J, respectively (10,14,20). The simultaneous deletion of both JBP1 and JBP2 yields a cell line that is unable to synthesize base J unless cells are fed hmU (14).…”

“…Deletion of either JBP1 or JBP2 from the bloodstream form T. brucei results in a 20-and 8-fold reduction in the levels of base J, respectively (10,14,20). The simultaneous deletion of both JBP1 and JBP2 yields a cell line that is unable to synthesize base J unless cells are fed hmU (14). Finally, using recombinant protein produced in Escherichia coli, we demonstrated that JBP1 stimulates the hydroxylation of thymidine in the context of dsDNA in a Fe 2ϩ , 2-oxoglutarate-, and O 2 -dependent manner (15).…”