Base J is synthesized in a two-step pathway. (A) Schematic model of the J-biosynthesis pathway. Step 1 involves the oxidation of thymidine (dT) residues in DNA forming the intermediate HOMedU. In step 2 the intermediate (HOMedU) is then glucosylated, by a glucosyl-transferase enzyme. This is then glucosylated, by a glucosyl-transferase enzyme, forming base J (dJ). The initial site-specific hydroxylation of thymidine, by TH, is thought to represent the key regulatory step of J biosynthesis. (B) Schematic diagram of the two proposed THs: JBP1 and JBP2. Shown in blue is the N-terminal region shared between both proteins. Within this region is the ∼70-amino-acid motif, indicated by the solid black box, which is related to the functional domain of members the Fe²⁺/2-oxoglutarate dependent hydroxylase family. Indicated above and below the putative TH motif (TH) for JBP2 and JBP1 is the amino acid signature shared among members of this hydroxylase family. The four key residues within this motif that have been implicated in catalysis by other members of this family of proteins (including JBP1), and which were mutated to alanine in JBP2 for the studies presented here, are highlighted in red. The C-terminal SWI2/SNF2 domain of JBP2 is shown in black.

Southern blot analysis reveals the deletion of both JBP1 and JBP2. (A) DNA was isolated from WT bloodstream cells (WT427), and cells from which either one (JBP2^−/+(H)) or both (JBP2^{−/− (H/H)}) alleles of JBP2 were deleted as previously described (9). JBP1 was then deleted from this cell line by by LOH (JBP2^{−/− (H/H)}/JBP1^{−/− (P/P)}) and by conventional methods (JBP2^{−/− (H/H)}/JBP1^{−/− (P/B)}). DNA samples were digested with PstI and probed for the presence of the JBP2 (left) or JBP1 (right) open reading frame (ORF). A clear loss of a band correlating with JBP2 and JBP1 is apparent in cell lines from which all four JBP alleles were deleted, regardless of the method of generation. The 1 kb DNA size marker is indicated on the left. The WT gene fragments are indicated by the arrows on the right. A cross-reactive 3 kb band is visible on the JBP1 ORF hybridization. H, P and B refer to the hygomycin, puromycin and blasticidin resistance genes, respectively. (B) Map demonstrating area of JBP1 or JBP2 targeted by deletion constructs. Pst I, HindIII, Nar I restriction sites are indicated. Puro, puromycin resistance gene; Hygro, hygromycin resistance gene. The ORF probes used in panel A are indicated by the lines.

Reexpression of JBP2 and JBP1 in J-null bloodstream cells stimulates site-specific J synthesis. In order to localize J within the genome, DNA was isolated from the indicated cell lines, sonicated and J containing DNA fragments immunoprecipitated using J antisera. 10% of the input DNA (IN) and 100% of the immunoprecipitated DNA (IP) was blotted onto nitrocellulose. Blots were then hybridized with radio-labeled DNA probes corresponding to indicate regions of the trypanosome genome. (A) The percent IP was calculated for each region of the genome analyzed for the indicated cell line and values were normalized to background hybridization. ENO, enolase; TEL, telomere. The corresponding repeat regions were referred by 177, 70 and 50. Each value represents a mean between three and five hybridizations ± SEM. (B) Representative auto-radiographs of the hybridization are shown.

Proposed mechanism of JBP1 and JBP2 catalyzed thymidine hydroxylation. The two-step J-biosynthesis pathway in T. brucei. Indicated is the proposed mechanism of JBP2/JBP1 catalyzed hydroxylation of thymidine residues during the initial step of the pathway, as discussed in the text. Bypassing Step 1 (and therefore JBP catalyzed thymidine hydroxylation) can be achieved through feeding cells HOMedU, as indicated. The absence of base J in the genome of the J null cell, but its ability to convert incorporated HOMedU to J, indicates that only Step 1 of the synthesis pathway is ablated upon deletion of JBP1 and JBP2. The glucosyl transferase (GT) enzyme is still active in this cell line.

Conserved residues with in the TH domain of JBP2 are critical for the protein to stimulate de novo J synthesis. (A) Dot-blot analysis of the JBP2 TH mutants. DNA was isolated from procyclic (insect stage) cells expressing either WT or mutant JBP2 and analyzed for J content by spotting DNA diluted onto a membrane and incubated with anti-J antisera. BS, WT bloodstream form cells; PC, WT procyclic cells; PC+JBP2, procyclic cells expressing JBP2 GFP; WT, procyclic cells expressing WT JBP2. H391A, D393A, H441A, R445A are procyclic cells expressing mutant versions of JBP2 as indicated in Figure 1. V459A is a control substitution of a residue outside the catalytic domain. (B) Expression of WT and mutant JBP2 in insect stage cells. Procyclic trypanosomes expressing either WT or mutant JBP2 were analyzed by western blot using anti-JBP2 antisera. Anti-La antisera was used as a loading control. Extract from untransfected procyclic cells is included as a negative control. (C) JBP2-GFP expression in procyclic trypanosomes was visualized microscopically by anti-GFP staining on formaldehyde fixed cells. DAPI staining reveals the localization of the nucleus (N) and the kinetoplast (K). WT JBP2, procyclic cells expressing WT protein; D391A, H441A are the corresponding mutant JBP2-GFP proteins.

JBP2 and JBP1 are essential for catalyzing the Step 1 of J biosynthesis in bloodstream trypanosomes. DNA was isolated from the indicated cell lines and analyzed for J content by anti-J DNA dot blot as in Figure 2. BS, WT bloodstream form cells; PC, procyclic cells; JBP2^−/−, JBP2 knock out bloodstream; JBP1^−/−, JBP1 knock out bloodstream; JBP2^−/−/JBP1^−/−, JBP2 and JBP1 knock out bloodstream; JBP2^−/−/JBP1^−/− + JBP2, JBP2 and JBP1 knock out bloodstream cells in which an ectopic JBP2 GFP is expressed; JBP2^−/−/JBP1^−/− + HOMedU, JBP2 and JBP1 knock out bloodstream cells feed with HOMedU; JBP2^−/−/JBP1^−/− + JBP2 + JBP1, JBP2 and JBP1 knock out bloodstream cells expressing both JBP2GFP and JBP1.

JBP2 stimulates HOMedU formation in vivo. (A) Specificity of the HOMedU antibody was evaluated by immunoprecipitation assays using ³²P-labeled mononucleotides derived from oligos containing HOMedU (HMU), base J (J) or thymidine (T) (see Methods section for oligo sequences). Postlabeled mononucleotides were incubated with α-HOMedU antibody conjugated to paramagnetic beads, washed to remove nonspecifically bound nucleotides followed by scintillation counting. Data are representative of three independent assays. Data are background subtracted and adjusted to results from unmodified DNA (T). (B) Anti-HOMedU immunoprecipitation analysis was used to examine HOMedU content of WT and the indicated mutant T. brucei cell lines. Genomic DNA was isolated from the indicated cell lines and analyzed for HOMedU content as in panel A. J-null (Null); J-null expressing WT JBP2GFP (Null+JBP2); J-null feed HOMedU (Null+HOMedU); J-null expressing JBP2 H441A mutant (Null+ JBP2 TH mutant). Each IP was repeated in triplicate and average ± SEM is shown.