Abstract
Switching of the Variant Surface Glycoprotein (VSG) in Trypanosoma brucei provides a crucial host immune evasion strategy that is catalysed both by transcription and recombination reactions, each operating within specialised telomeric VSG expression sites (ES). VSG switching is likely triggered by events focused on the single actively transcribed ES, from a repertoire of around 15, but the nature of such events is unclear. Here we show that RNA-DNA hybrids, called R-loops, form preferentially within sequences termed the 70 bp repeats in the actively transcribed ES, but spread throughout the active and inactive ES, in the absence of RNase H1, which degrades R-loops. Loss of RNase H1 also leads to increased levels of VSG coat switching and replication-associated genome damage, some of which accumulates within the active ES. This work indicates VSG ES architecture elicits R-loop formation, and that these RNA-DNA hybrids connect T. brucei immune evasion by transcription and recombination.
Highlights
The genome provides the blueprint for life and is normally protected from rapid content change by high fidelity DNA replication and a range of repair pathways
A key event in antigenic variation is the initiation of the change in expression of the surface protein gene, though how this occurs has been detailed in very few pathogens
We show that R-loops accumulate throughout all Variant Surface Glycoprotein (VSG) expression sites (ES) in the absence of the RNase H enzyme, indicating RNA-DNA hybrids form in these transcription sites and are normally resolved by removing the RNA
Summary
The genome provides the blueprint for life and is normally protected from rapid content change by high fidelity DNA replication and a range of repair pathways. Enhanced genome change is more localised and caused by deliberate lesion generation, such as during yeast mating type switching, which is induced by HO endonuclease-mediated cleavage in Saccharomyces cerevisiae [4] and locus-directed replication stalling in Schizosaccharomyces pombe [5]. Antigenic variation is a very widespread pathogen survival strategy, involving stochastic switches in surface antigens to thwart host adaptive immunity [8], and locus-directed gene rearrangement is a common route for the differing reactions used in bacteria, fungi and protists [9, 10]. Only in the bacteria Neisseria gonorrhoeae is initiation well understood; here, HR catalyses movement of silent, non-functional pilS genes into a pilE expression locus via transcription-induced guanine quadruplex formation [11]. In no other pathogen has the initiation event(s) during antigenic variation been resolved in this detail
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