The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

Abstract

Trypanosomatids contain an unusual DNA base J (β-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe2+ and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe2+ and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.

JBP1 and JBP2 Proteins Are Fe2+/2-Oxoglutarate-dependent Dioxygenases Regulating Hydroxylation of Thymidine Residues in Trypanosome DNA

“…The two highly conserved histidine metal binding ligands in the core jelly roll domain of Fe 2ϩ /2-OG enzymes have been shown to be essential for Fe 2ϩ binding and hydroxylase function (12,17). Consistent with this finding, mutation of residues H189A and H239A in JBP1 ablated J synthesis in vivo (8). We see here that in vitro Fe 2ϩ binding and thymidine hydroxylation is ablated in the H189A/H239A mutant, demonstrating that Fe 2ϩ binding is critical for TH activity of JBP1 (Fig.…”

“…The recent identification and characterization of the TET enzymes catalyzing the hydroxylation of 5-MeC in DNA strengthened the identification of JBPs as dioxygenases (11), characterizing them as members of the new TET/JBP subfamily of Fe 2ϩ /2-OG dioxygenase (10,11). However, no direct analysis of JBP thymidine hydroxylation has been demonstrated despite a clear in vivo gene knock out and mutagenesis studies demonstrating the importance of JBP1 and JBP2 in J biosynthesis (8,9,30). The oxidation of thymidine residues by a thymidine hydroxylase is unusual.…”

“…Preparation of Recombinant JBP1-Recombinant His-tagged L. tarentolae JBP1 (Lt-JBP1) was produced essentially as previously described (8) with the following modifications. The Lt-JBP1 vector was transformed to BL21-DE3 T1 R (21).…”

“…12). Mutation of these conserved residues within the TH domain of JBP1 and JBP2 * This work was supported, in whole or in part, by National Institutes of Health ablates enzyme function in J synthesis in vivo, supporting their classification as Fe 2ϩ /2-OG dioxygenases (8,9). Deletion of either JBP1 or JBP2 from bloodstream form T. brucei resulted in a 20-and 8-fold reduction in J levels, respectively (13,14).…”

“…Although the glucosyltransferase has not been identified, two proteins involved in the first step (JBP1 and JBP2) (6, 7) have been characterized. Both JBP1 and JBP2 (8,9) contain a putative TH domain at the N terminus that has led to the designation of these enzymes belonging to the new TET/ JBP subfamily of dioxygenases that require Fe 2ϩ and 2-oxoglutarate (2-OG) for activity (10,11). Family members are typically identified on a structural level by the presence of a jelly roll ␤-helix sheet that contains four key conserved residues involved in the binding of Fe 2ϩ and 2-OG and are essential for catalytic activity (see for review, see Ref.…”