RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation inTrypanosoma brucei

Abstract

AbstractRNA-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 asTrypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialised telomeric locus that directs transcription of just oneVSGfrom thousands. How suchVSGrecombination is targeted and initiated is unclear. Here, we show that a key enzyme ofT. bruceihomologous 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 transcribedVSG, and that mutation of RAD51 alters RNA-DNA hybrid abundance both around the transcribedVSGand across the silentVSGarchive. 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 criticalT. bruceiimmune evasion reaction of antigenic variation.