Discussion

Key to host immune evasion is antigenic variation in the bloodstream, triggered by subtelomeric DNA-breaks adjacent to the single active Variant Surface Glycoprotein (VSG) gene. In mammalian cells, distinct single-stranded DNA (ssDNA)-binding proteins control DNA-recombination and repair at different loci and at different cell-cycle stages by protecting ssDNA and recruiting other repair-associated factors to sites of DNA-damage. Mammalian replication protein A (RPA), for example, is a conserved ssDNA binding-protein, which coats ssDNA in a cell-cycle dependent manner in the S- and G2-phases. We have used Trypanosoma brucei RPA DNA-damage associated foci to monitor meganuclease-induced DNA breaks at distinct loci. RPA-foci are restricted to the nucleolus following a break within ribosomal DNA, but are found at extranucleolar sites following breaks at active or silent VSG loci. Telomeres and active and silent VSG damage-foci distribute towards the nuclear interior in bloodstream-form T. brucei but tend towards the nuclear periphery in the insect-stage. Thus, DNA damage in T. brucei appears to generate ssDNA at all loci tested. Most strikingly though, we found that RPA-foci were readily detected at all cell-cycle stages; foci formed in S-phase and persisted post-mitosis and beyond, even while histone gH2A DNA-repair foci were disassembled. Our findings reveal a ‘divide and repair later’ strategy, whereby DNA-resection is not linked to a cell cycle checkpoint in T. brucei. This likely suits the parasitic life-style and facilitates the generation of genetic diversity within subtelomeric domains.