Transcript Piet Borst
The discovery of a new base in DNA, base J In 1978 we started a study of the mechanism of antigenic variation in African trypanosomes. These trypanosomes are covered with a dense layer of a single protein, the variant surface glycoprotein (VSG), and they are unique in multiplying freely in the bloodstream of the mammalian host. They escape from the host’s immune system by regularly switching their coat. In the 80s we discovered how the trypanosome manages to produces innumerable different coats (1). The VSG genes are present in telomeric expression sites. The trypanosome can switch the VSG gene expressed either by replacing the active gene in the expression site by a different gene (the major mechanism), or by switching from one expression site to another one (2). In 1983 my student, André Bernards, noted something funny when an expression site was switched off: there were a few specific sites in the inactive expression site that had become partially uncleavable by restriction endonucleases (3). This suggested that silencing was accompanied, even in this primitive uni-cellular eukaryote, by DNA modification. Finding the DNA modification involved in the control of antigenic variation looked like a sexy topic to me, but not to my collaborators. They expected it to be methyl-C again, and, moreover, the unusual base was present in low amounts and therefore hard to find. Fortunately the technician of Ronald Plasterk, at that time another PI working on the same floor as I did, got interested. If I would take her as a graduate student, she would find the mystery base for me. At that time it had just become possible for technicians to get a PhD and she was the first in the Netherlands Cancer Institute to embark on this risky trajectory. I took her and the rest is history. The base she identified in four years was not only sexy, it was an entirely new base in DNA and moreover the first hypermodified base in eukaryotic DNA, beta-glucosylhydroxymethyluracil (4). We called it base J after the student’s first name, Jeanette Gommers-Ampt, shown here with a model of Jnucleoside. The elucidation of the biosynthesis and functioning of base J took much longer than four years: ten years to find the enzymes involved in the biosynthesis of base J, the hydroxylases that oxidize selected T’s in DNA (5) and another ten years to discover a function of base J. In both cases the end results were rewarding. The hydroxylases induced L. Aravind to look for analogous enzymes in mammals and he found the TET enzymes, oxidizing methylC; and base J turned out to act as a terminator of Pol II leading to spectacular readthrough transcription when we removed J (6). Source: Piet Borst (NKI) References (1) Hoeijmakers J.H.J., Frasch A.C.C., Bernards A., Borst P., Cross G.A.M., 1980 Novel expression-linked copies of the genes for variant surface antigens in trypanosomes, Nature, 284: 78-80. (2) Bernards A., De Lange T., Michels P.A.M., Huisman M.J., Borst P., 1984 Two models of activation of a single surface antigen gene of Trypanosoma brucei, Cell, 36: 163-170. (3) Bernards A., Van Harten-Loosbroek N., Borst P., 1984 Modification of telomeric DNA in Trypanosoma brucei, a role in antigenic variation?, Nucl. Acids Res., 12: 4153-4170. (4) Gommers-Ampt J.H., Van Leeuwen F., De Beer A.L.J., Vliegenhart F.G., Dizdaroglu M., Kowalak J.A., Crain P.F., Borst P., 1993 β-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan Trypanosoma brucei. Cell,75: 1129-1136. (5) Yu Z., Genest P.A., Ter Riet B. Sweeney K., Dipaulo C., Kieft R., Christodoulou E., Perrakis A., Simmons J., Hausinger R., Van Luenen H., Rigden D., Sabatini R., Borst P., 2007 The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase, Nucl. Acids Res. 35: 2107-2115. (6) Van Luenen H., Farris C., Jan S., Genest P.A., Tripathi P., Velds A., Kerkhoven R.M., Nieuwland M., Haydock A., Ramasamy G., Vainio S., Heidebrecht T., Perrakis A., Pagie L., Van Steensel B., Myler P., Borst P., 2012 Glucosylated hydroxymethyluracil (DNA base J) prevents transcriptional read-through in Leishmania, Cell, 150(5): 909-921.