TOPO3alpha influences antigenic variation by monitoring expression-site-associated VSG switching in Trypanosoma brucei

Homologous recombination (HR) mediates one of the major mechanisms of trypanosome antigenic variation by placing a different variant surface glycoprotein (VSG) gene under the control of the active expression site (ES). It is believed that the majority of VSG switching events occur by duplicative gene conversion, but only a few DNA repair genes that are central to HR have been assigned a role in this process. Gene conversion events that are associated with crossover are rarely seen in VSG switching, similar to mitotic HR. In other organisms, TOPO3alpha (Top3 in yeasts), a type IA topoisomerase, is part of a complex that is involved in the suppression of crossovers. We therefore asked whether a related mechanism might suppress VSG recombination. Using a set of reliable recombination and switching assays that could score individual switching mechanisms, we discovered that TOPO3alpha function is conserved in Trypanosoma brucei and that TOPO3alpha plays a critical role in antigenic switching. Switching frequency increased 10-40-fold in the absence of TOPO3alpha and this hyper-switching phenotype required RAD51. Moreover, the preference of 70-bp repeats for VSG recombination was mitigated, while homology regions elsewhere in ES were highly favored, in the absence of TOPO3alpha. Our data suggest that TOPO3alpha may remove undesirable recombination intermediates constantly arising between active and silent ESs, thereby balancing ES integrity against VSG recombination.