Enzymatic Formation of the Hypermodified DNA Base J (β‑D‑Glucopyranosyloxymethyluracil)
Robert Sabatini, Laura Cliffe, Saara Vainio and Piet Borst
Base J (β‑D‑glucopyranosyloxymethyluracil) is the only hyper‑modified DNA base known in eukaryotes. It is present in the nuclear DNA of all flagellated protozoa of the order of the Kinetoplastida and in the closely related unicellular alga Euglena gracilis. Base J is a minor constituent of DNA, replacing at most 1% of thymidines and it is mainly present in repetitive sequences, invariably including the telomeric repeats. The synthesis of the base involves two enzymatic steps: hydroxylation of a thymidine residue in DNA producing HOMedU in DNA as a free intermediate, followed by addition of the glucose moiety. The enzymes involved in J biosynthesis, thymidine hydroxylase and glucosyl transferase, represent novel enzymes. Base J was originally identified in Trypanosoma brucei based on its developmentally regulated synthesis and localization correlating with the silencing of telomeric surface antigen genes of this deadly human parasite. In this chapter, we will focus primarily on T. brucei, in which the majority of work on J has been carried out and a potential function for the modified base is evident. The early history of base J discovery and recent developments in our understanding of J biosynthesis and function have been reviewed in detail. This chapter highlights the methods to detect J and HOMeUra, our current knowledge of the regulation of base J synthesis during the parasite‘s lifecycle and our most recent attempts to define the elusive function of base J.