JBP1 and JBP2 are two distinct thymidine hydroxylases involved in J biosynthesis in genomic DNA of African trypanosomes.
Genomic DNA of African trypanosomes contains a hypermodified thymidine residue termed base J (beta-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 Fe(2+)/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.
Cliffe
Laura J
LJ
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Kieft
Rudo
R
Southern
Timothy
T
Birkeland
Shanda R
SR
Marshall
Marion
M
Sweeney
Kate
K
Sabatini
Robert
R
D000818:Animals; D002460:Cell Line; D016054:DNA, Protozoan / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018503:Genome, Protozoan; D005960:Glucosides / Q000096:biosynthesis* / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014498:Uracil / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry
D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D005960:Glucosides; C505199:J-binding protein, Trypanosoma brucei; D015800:Protozoan Proteins; C084880:5-((glucopyranosyloxy)methyl)uracil; D014498:Uracil; D006899:Mixed Function Oxygenases; C023162:thymidine,2-oxoglutarate dioxygenase