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.

Figure 1.

An experimental system to study end-joining. (A) The schematic maps illustrate the Tb11.02.2110 alleles in wild-type (WT), R^sP_a and RΔ strains. The meganuclease cleavage site is embedded within a dsRed Fluorescent Protein (RFP)–Puromycin ACetyltransferase (PAC) fusion gene. B, Bsp120I; H, HindIII; X, XcmI. (B). The RΔ strain was validated by Southern blotting with WT and R^sP_a controls. The RΔ and R^sP_a strains were grown in the absence or presence of tetracycline (1 µg ml⁻¹) for 1 week. Genomic DNA was digested with Bsp120I and HindIII. Bands representing the 2110 alleles are indicated to the right. In the R^sP_a strain, allelic HR regenerates the 6 kb allele while, in the RΔ strain, ectopic HR and end joining generate allele a fragments at 7.9 and 5.2 kbp, respectively; see fragment sizes in (A) and (C). (C) The schematic maps illustrate the result of ectopic HR and end joining expected to predominate in R^sP_a/Δ2110_b survivors. The TUB sequences flanking the R^sP cassette promote R^sP pre-mRNA trans-splicing and polyadenylation and also allow ectopic HR which replaces R^sP with an αTUB gene copied from chromosome 1.

Figure 2.

MMEJ is common in RΔ survivors. (A) As expected, the RΔ strain displays a reduced cloning efficiency after DSBR due to cell death after allelic HR. Data derived from dilution cloning in 96-well plates: −Tet, n = 4; +Tet, n = 6. (B) RΔ survivors display ectopic HR, one-sided MMEJ and MMEJ-based deletions as determined by DNA sequencing; n = 110.

Figure 3.

The distribution of microhomologies used for repair. (A) Frequency of microhomologies was mapped in 50-bp intervals on either side of the DSB. (B) Frequency of paired microhomologies was mapped as in A. All microhomology (MH) classes represented by >1 junction are indicated; see Table 1. (C) A PCR assay indicates a similar pattern of MMEJ at different loci across the genome. Products corresponding to frequent MH pairings and size of deletion are indicated; see Table 1.

Figure 4.

Microhomology classes; see Table 1 for more details. (A) MMEJ junctions (sense strand only) are shown for all 14 MH classes identified. The parental RFP (R) and PAC (P) sequences are shown above and below, respectively. The template switch site is indicated with white lettering. The junction types are as follows: X, processed within the largest homology patch; x, processed within a ‘minor’ homology patch; [x] processed outside the homology patches. Microhomologies are highlighted (≥3 bp patches plus 2 bp patches if within 1 bp of a larger patch). Sequence traces are shown for [x] junctions, 3X/x, 8X and all MH4 sub-classes (see the text), and the numbers of each sub-class recorded is indicated to the right. Asterisk at MH3, 5, 6 and 9 indicates the I-SceI cleaved terminus. (B) GC-content is over-represented within microhomology patches. *P < 0.0001 as determined using a χ² test.

Figure 5.

One-sided MMEJ-based gene conversion. (A) The schematic map illustrates four allelic one-sided MMEJ events; the gray box indicates HR and the lines indicate the locations of microhomologies, in the aldolase (ALD) processing sequence (cross-hatched box) linked to the NPT gene in this case. In clones 24 and 73, the terminal 21 codons of the 2110 gene are replaced with 11 new codons and a stop codon; presumably allowing the expression of a functional protein. (B) PCR amplification of fragments spanning the gene conversion tract reveals a product of the expected size in each case; wild-type (wt) cells serve as a control. The locations of the primers are indicated in A (small arrows). (C) The four one-sided allelic MMEJ junction sequences are shown; other details as in Figure 4A.

Figure 6.

One-sided MMEJ-based gene conversion is RAD51 dependent. (A) PCR assays indicate a similar pattern of MMEJ-based deletion in RAD51 and rad51 null strains (upper panel) while one-sided MMEJ-based gene conversion is ablated in rad51 null strains (lower panel). The locations of the primers are indicated in B. (B) The schematic map illustrates three ectopic one-sided MMEJ events. (i) and (ii) are from Figure 6A and (iii) is from (6); other details as in Figure 5A. (C) The three one-sided ectopic MMEJ junction sequences are shown; other details as in Figure 4A.