MOLECULAR MICROBIOLOGY A key event in survival Dave Barry and Richard McCulloch The parasitic microorganism Trypanosoma brucei evades recognition by its host’s immune system by repeatedly changing its surface coat. The switch in coat follows a risky route, though: DNA break and repair. VSG genes that lie at the ends (telomeres) of a set of mini-chromosomes, as well as a further 1,600 silent genes — of which two-thirds are pseudogenes — on the main chromosomes 4 . The potential for mosaic variation therefore seems beyond estimation. Intact archival genes are duplicated starting from an upstream set of repeat sequences each 70 base pairs (bp) long 5 , all the way to sequences at the down- stream end of the coding sequence, or, in the case of silent telomeric genes, perhaps to the nearby end of the chromosome. As gene con- version in other organisms is initiated by a DSB in the conversion site, such a break has been proposed also to occur in the T. brucei VSG saving a critical intermediate from degradation. The penultimate reaction of the sequence, in which the phosphate is attached to the nucleo- side, is another beautiful example of the influence of systems chemistry in this set 2 of interlinked reactions. The phosphorylation is facilitated by the presence of urea 4 ; the urea comes from the phosphate-catalysed hydrolysis of a by-product from an earlier reaction in the sequence. The authors wrap up their synthetic tour de force by using ultraviolet light to clean up the reaction mixture. They report that ultra- violet irradiation destroys side products while simultaneously converting some of the desired ribocytidine product to ribouridine (the second pyrimidine component of RNA). The development of this complex photochemistry required remarkable mechanistic insight from Powner and colleagues, who not only correctly predicted that ultraviolet irradiation would destroy the majority of the by-products, but also that the desired ribonucleotides would withstand such treatment. The authors’ careful study 2 of every poten- tially relevant reaction and side reaction in their sequence is a model of how to develop the fundamental chemical understanding required for a reasoned approach to prebiotic chem- istry. By working out a sequence of efficient reactions, they have set the stage for a more fruitful investigation of geochemical scenarios compatible with the origin of life. Of course, much remains to be done. We must now try to determine how the various starting materials could have accumulated in a relatively pure and concentrated form in local environments on early Earth. Furthermore, although Powner and colleagues’ synthetic sequence yields the pyrimidine ribonucleotides, it cannot explain how purine ribonucleotides (which incorporate guanine and adenine) might have formed. But it is precisely because this work opens up so many new directions for research that it will stand for years as one of the great advances in prebiotic chemistry. ■ Jack W. Szostak is in the Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. e-mail: szostak@molbio.mgh.harvard.edu 1. Joyce, G. F. & Orgel, L. E. in The RNA World (eds Gesteland, R. F., Cech, T. R. & Atkins, J. F.) 23–56 (Cold Spring Harbor Laboratory Press, 2006). 2. Powner, M. W., Gerland, B. & Sutherland, J. D. Nature 459, 239–242 (2009). 3. Orgel, L. E. Crit. Rev. Biochem. Mol. Biol. 39, 99–123 (2004). 4. Lohrmann, R. & Orgel, L. E. Science 171, 490–494 (1971). Like many other single-celled pathogens, the protozoan Trypanosoma brucei, which causes African sleeping sickness in humans, undergoes antigenic variation — that is, it periodically switches its variant surface glyco- protein (VSG), the molecule targeted by host antibodies. But how switching is triggered has remained largely elusive. On page 278 of this issue, Boothroyd et al. 1 show that a DNA double-strand break (DSB) upstream of the T. brucei VSG gene is the likely primary event in this process. Their results add to the few, albeit crucial, cases in which DSBs trigger develop- mental processes: these include mating-type switching in yeast, rearrangements of immune- system genes in humans and meiotic cell division to produce sex germ cells 2 . Antigenic switching can occur through several genetic strategies, the most common being the differential activation of an archive of silent genes and pseudogenes. Although only one gene is transcribed, from a specialized expression site, switching occurs when silent genes, or their fragments, are duplicated in the expression site by a gene-conversion process, replacing all or part of the expressed gene. In some pathogens, the expressed gene can be constructed as a mosaic from several archival pseudogenes; such a combinatorial strategy expands the scale of variation enormously, with, for example, five pseudogenes giving rise to hundreds of combinations 3 . Trypanosoma brucei has evolved an even more staggeringly complex system. It, too, transcribes a single VSG gene, but the sources of sequences that contribute to switching are large and diverse. It has several inactive expres- sion sites, and its archive contains up to 200 Gene promoter 70-bp repeats Not detected 83% 17% Endonuclease Switch by repair Expression sites 5–15 VSG genes Arrays ~1,600 VSG genes Mini-chromosomes <200 VSG genes c a b Transcribed VSG gene DSB Figure 1 | Antigenic switching and sources. Boothroyd et al. 1 used an endonuclease enzyme to induce a DNA double-strand break (DSB) adjacent to the 70-bp-repeat region of the active VSG gene in Trypanosoma brucei. Consequently, the region from the DSB site to the end of the VSG gene was deleted. The protozoan filled this gap by a repair process, using silent VSG loci on other chromosomes as template. Locations of donor sequences included: (a) expression sites (of which there are 5–15 per strain) at the telomeres of the main chromosomes; (b) telomeres of some 100 mini-chromosomes found in the T. brucei genome; and (c) tandemly arrayed VSG genes in the main chromosomes. The copied regions stretched from the 70-bp-repeat regions to the telomere, or, for intact genes, to the end of the VSG. The frequencies of conversions the authors detected (shown as percentages) differ from those observed during infections with natural strains of T. brucei, in which mini-chromosomes dominate as donors. Brackets denote the duplicated region, with dashed sections indicating uncertainty over where the duplication ends. Broad arrows indicate genes; narrow arrows, repetitive DNA sequences (70-bp repeats are shown in black and white). Coloured arrows are different VSG genes; grey arrows, genes other than VSG. 172 NATURE|Vol 459|14 May 2009 NEWS & VIEWS © 2009 Macmillan Publishers Limited. All rights reserved