Proposed pathway for J biosynthesis. First, a thymidine hydroxylase catalyzes the formation in DNA of HMdU. Second, HMdU in DNA is converted into J by a putative glucosyl transferase.

Localization of mutant Lt GFP-JBP1 in JBP1 null trypanosomes. JBP1 null trypanosomes (JBP1 dKO) were transfected with the wild-type (WT) or the mutant (H189A, D191A, H239A, R255A) Lt GFP-JBP1 fusions, DAPI-stained and visualized live on agarose slides by microscopy. DAPI staining shows the location of the nucleus (N) and of the kinetoplast (mitochondrial DNA; K).

Determination of J levels in the DNA of JBP1 null trypanosomes expressing the Lt JBP1 mutants. (A) Genomic DNA of wild-type T. brucei bloodstream form 427 line (WT), JBP1 null trypanosomes (JBP1 dKO) and JBP1 null trypanosomes expressing the wild-type Tb GFP-JBP1 fusion (Tb JBP1 WT), the wild-type Lt GFP-JBP1 fusion (Lt JBP1 WT) or the mutant Lt GFP-JBP1 fusions (H189A, D191A, H239A, R255A, V259A) were serially diluted in steps of two, denatured, spotted on a nitrocellulose membrane and incubated with a polyclonal J antiserum (left panel). The membrane was hybridized with a tubulin DNA probe as a loading control (right panel). (B) Western blot on lysates of the JBP1 null trypanosomes (JBP1 dKO) and the JBP1 null trypanosomes expressing the wild-type (WT) and the mutant (H189A, D191A, H239A, R255A, V259A) Lt GFP-JBP1 fusions using an Lt JBP1 antiserum.

Determination of the J-binding activity of purified Lt JBP1 mutants by electromobility shift assay. (A) Upper panel: Electromobility shift assay with the purified His-tagged wild-type (WT) and mutant (H189A, D191A, H239A, R255A, V259A) Lt JBP1 proteins. Proteins (10 fmol) were incubated with a radioactively labeled double-stranded oligonucleotide containing J (J-DNA) (10 fmol) and run on 4.5% native acrylamide gel. The gel was dried and the signal detected by autoradiography. The bands corresponding to the J-DNA/Lt JBP1 and the free J-DNA are indicated. Lower panel: Segment of a Coomassie-brilliant-blue-stained acrylamide gel with the His-tagged wild-type (WT) and mutant (H189A, D191A, H239A, R255A, V259A) Lt JBP1 proteins purified from E. coli. Only the segment of the gel with the His-tagged Lt JBP1 band is shown. (B) Example of a determination of the J-binding activity of mutant Lt JBP1 by electromobility shift assay. His-tagged wild-type (WT) and H189A + D191A mutant Lt JBP1 proteins were purified from E. coli and incubated with a radioactively labeled double-stranded oligonucleotide containing J (J) or lacking J (T) in the presence or absence of a Lt JBP1 antibody (α-Lt JBP1). Samples were run on a 4.5% native acrylamide gel. The gel was dried and the signal detected by autoradiography. Lanes without recombinant protein are indicated by ‘–’. The position of the free DNA, the J-DNA/Lt JBP1 complex and the J-DNA/Lt JBP1 + α-Lt JBP1 complex is indicated.

Sequence alignment of the conserved all-β core (see text) of JBP1 and JBP2 sequences with diverse members, of known structure, from the Fe²⁺- and 2-oxoglutarate-dependent dioxygenase superfamily. JBP1 and JBP2 sequences are given with species as follows: Tb, T. brucei; Tc, T. cruzi; Lt, L. tarentolae; Lm, L. mexicana and Cf, Crithidia fasciculata. The gi codes in the nr database (Wheeler et al., 2005) are: 62361410 for Lt JBP1, 68124616 for Lm JBP1, 6018041 for Tb JBP1, 71421637 for Tc JBP1, 6018043 for Cf JBP1, 68125217 for Lm JBP2, 71750205 for Tb JBP2 and 71422266 for Tc JBP2. The dioxygenase structures are as follows: DCS, deacetoxycephalosporin C synthase (PDB code 1dcs) (47); AS, anthocyanidin synthase (1gp6); IS, isopenicillin N synthase (1obn); TD, taurine dioxygenase (1os7); FIH, factor inhibiting HIF (1iz3); PH, proline 3-hydroxylase (1e5s); QD, quercetin 2,3-dioxygenase (1y3t). The better conserved positions within the conserved core are shown in bold font with invariant positions additionally italicized. Numbers in the alignment represent the positions of the amino acids in Lt JBP1 and mark the positions of large deletions that are not shown for clarity. The predicted secondary structure of Lt JBP1 is shown above the alignment and the actual secondary structure of quercetin 2,3-dioxygenase (PDB code 1y3t) is shown below the alignment. The long arrows represent β-sheets and the tubes α-helices. Arrowheads below the alignment indicate the four key residues, largely conserved throughout the Fe²⁺- and 2-oxoglutarate-dependent dioxygenase superfamily that bind the iron (His, Asp) or 2-oxoglutarate (Arg) (23,24). The residues of Lt JBP1 that were mutated to alanines: H189, D191, H239, R255, V259, are shown by the arrowheads above the alignment. Note that there are near identical pairs of sequences for both JBP1 and JBP2 in the T. cruzi genome: only one of each pair is shown in the alignment.