Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

Abstract

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue.

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

“…Sequence alignments reveal that the parasite RAD51 homologs share between 60 to 80% sequence identity with human RAD51 and that sequence conservation appears to extend outside the RecA fold, as an HhH and a PM motif have been reported in TbRad51 (179)(180)(181). Genetic analysis in T. brucei revealed that Rad51 Ϫ/Ϫ mutants display increased sensitivity to DNA-damaging agents, such as methyl methanesulfonate (MMS) and phleomycin, and reduced integration of transformed DNA, supporting a role in homologous recombination (113,179,182). In vivo, RAD51 is known to assemble at DSB sites, where it forms discrete nuclear foci after DNA damage (183,184).…”

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…Indeed, though this switch reaction does not involve DNA rearrangements, it remains possible that it shares repair-related or replication-related functions that act in recombination-based switching (see below). Such a link would explain why mutation of core homologous recombination (HR) factors not only impairs VSG switching by recombination (see below), but also VSG switching by transcription (108)(109)(110), and why multiple treatments that cause DNA damage or replication arrest in T. brucei can elevate silent BES expression (111).…”

“…5) have been limited to understanding the activation of intact VSGs (106,108,110,114,117,(127)(128)(129). This stems from the fact that these studies have used in vivo or in vitro selection strategies that detect the most frequently activated VSGs; the lack of detectable mosaic VSG formation in these studies may suggest that SGC is less efficient than intact VSG conversion, though a mechanistic switch during an infection cannot be currently ruled out.…”

“…The numbers of Rad51 paralogs vary widely in eukaryotes, with five in mammals but only one in Caenorhabditis elegans. Among single-celled eukaryotes, trypanosomes appear to possess a relatively large repertoire of four proteins (108,144). In T. brucei each RAD51 paralog acts in DNA recombination and repair.…”

“…Multiple lines of evidence support such reaction flexibility. First, mutation of RAD51-dependent HR impairs, but does not abolish, VSG recombination (108)(109)(110)157). Second, though the 70-bp repeats make an important contribution to VSG switching, their removal is not an impediment (114,127).…”