Mapping of double-stranded DNA breaks in Trypanosoma brucei using BLISS

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis and centromere function. Increasing evidence suggests RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: they can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialised telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome-wide co-localisation with DNA breaks, and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG, and that mutation of RAD51 alters RNA-DNA hybrid abundance both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.