title: Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.
abstract: Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States.
authors.forename: Whitney
authors.initials: W
authors.lastname: Bullard
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States.
authors.forename: Laura
authors.initials: L
authors.lastname: Cliffe
authors.affiliation: Environmental Toxicology Graduate Program, United States.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Environmental Toxicology Graduate Program, United States; Department of Chemistry, University of California, Riverside, CA, United States.
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, United States. Electronic address: rsabatini@bmb.uga.edu.
authors.forename: Robert
authors.initials: R
authors.lastname: Sabatini
mesh_terms: D016054:DNA, Protozoan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007891:Leishmania / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010432:Pentoxyl / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013941:Thymine / Q000737:chemistry / Q000378:metabolism
chemical_list: D016054:DNA, Protozoan; D015800:Protozoan Proteins; C008055:5-hydroxymethyluracil; D010432:Pentoxyl; D005964:Glucosyltransferases; D013941:Thymine
keywords: Base J; Epigenetic; Glucosyltransferase; RNA polymerase II transcription; Trypanosomatid
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