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Microhomology-mediated deletion and gene conversion in African trypanosomes.

Glover L, Jun J, Horn D - Nucleic Acids Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

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.

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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.
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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.

Mentions: aJunction types are defined in the legend to Figure 4.


Microhomology-mediated deletion and gene conversion in African trypanosomes.

Glover L, Jun J, Horn D - Nucleic Acids Res. (2010)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045614&req=5

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.
Mentions: aJunction types are defined in the legend to Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

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.

Show MeSH
Related in: MedlinePlus