Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA

•Only specific DNA sequences can promote base J synthesis in vivo.•Base J associated glucosyltransferase does not possess DNA sequence specificity.•Specific localization of base J throughout the genome is likely not due to the glucosyltransferase catalyzing the second step of base J synthesis.

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.

Graphical abstractIn vivo only particular DNA sequences can become J modified. The specificity of J modification is not due to the JGT.Figure optionsDownload full-size imageDownload high-quality image (119 K)Download as PowerPoint slide