Microhomology-mediated deletion and gene conversion in African trypanosomes

Abstract

Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in individual DSBR survivors exclusively revealed MMEJ-based deletions but no NHEJ. A survey of microhomologies typically revealed sequences of between 5 and 20 bp in length with several mismatches tolerated in longer stretches. Mean deletions were of 54 bp on the side closest to the break and 284 bp in total. Break proximity, microhomology length and GC-content all favored repair and the pattern of MMEJ described above was similar at several different loci across the genome. We also identified interchromosomal gene conversion involving HR and MMEJ at different ends of a duplicated sequence. While MMEJ-based deletions were RAD51-independent, one-sided MMEJ was RAD51 dependent. Thus, we describe the features of MMEJ in Trypanosoma brucei, which is analogous to micro single-strand annealing; and RAD51 dependent, one-sided MMEJ. We discuss the contribution of MMEJ pathways to genome evolution, subtelomere recombination and antigenic variation.

Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos

“…However, in vitro studies in human cells indicate that DNA Lig3 and Lig1 are required for MMEJ [5,7,60]. MMEJ was also considered to be a type of SSA, called micro-SSA, because both repair DNA homologous ends result in a repeat sequence deletion [61,62]. In our study, we utilized different tools (morpholinos, dominant-negative proteins, mutants) and different approaches (endogenous DSBs and exogenous vectors) to study the relative roles of Lig4 and Lig3 in MMEJ repair in zebrafish embryos.…”

DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

“…Nonetheless, current evidence suggests that SSA is not a major mechanism of DNA repair in Plasmodium (13). For another eukaryotic pathogen, T. brucei, chromosomal and in vitro plasmid EJ assays produce only MMEJ-generated repair products (102)(103)(104). NHEJ is not used, and T. brucei Ku functions only in telomere maintenance (105).…”

DNA Recombination Strategies During Antigenic Variation in the African Trypanosome

“…In contrast, MMEJ is readily detected in T. brucei, and may have assumed the role of NHEJ in DSB repair (165)(166)(167)(168). Testing whether MMEJ provides a route for VSG gene conversion in the absence of RAD51-dependent HR is hampered by the lack of clarity in the machinery involved, but it is increasingly clear that this reaction is highly flexible, for instance contributing to many forms of structural genome variation and acting in immunoglobulin rearrangements (161).…”

“…Classical HR in T. brucei seems too demanding to explain this process, being relatively intolerant of base mismatches and requiring extensive regions of homology (157). The RAD51-independent MMEJ pathway, requiring only ∼5 to 15 bp of homology and tolerant of mismatches (166)(167)(168), appears a more promising candidate, but the machinery involved in this process is unknown. Just as importantly, it is unclear if the form of strand transfer and break repair in MMEJ is capable of catalyzing VSG SGC; for instance, are mosaic VSGs generated in a stepwise fashion (perhaps over a number of cell cycles) involving successive gene conversions (37), or can they result from a single construction event?…”