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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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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.
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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.

Mentions: We employed clonogenic assays to determine the proportion of RΔ cells that survive I-SceI-induced lesions (Figure 2A). The RsPa strain served as a control for this analysis and, consistent with previous findings, indicated >50% survival. In contrast, and consistent with cell death following allelic HR, the RΔ strain displayed <10% survival (Figure 2A). To distinguish between different repair pathways and to quantify the relative contribution of each pathway in RΔ cells, we derived a panel of survivor clones. This approach was favored over analysis of mixed populations because DNA amplification is required to access the sequence of end-joining junctions (see below) and amplification of multiple, related DNA fragments is prone to ‘template-switching’ artifacts and amplification bias. In addition, the cloned-survivor approach improves the chance of revealing repair junctions in unanticipated locations since clones that initially fail to reveal a junction can be specifically targeted for further analysis.Figure 2.


Microhomology-mediated deletion and gene conversion in African trypanosomes.

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

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

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

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.
Mentions: We employed clonogenic assays to determine the proportion of RΔ cells that survive I-SceI-induced lesions (Figure 2A). The RsPa strain served as a control for this analysis and, consistent with previous findings, indicated >50% survival. In contrast, and consistent with cell death following allelic HR, the RΔ strain displayed <10% survival (Figure 2A). To distinguish between different repair pathways and to quantify the relative contribution of each pathway in RΔ cells, we derived a panel of survivor clones. This approach was favored over analysis of mixed populations because DNA amplification is required to access the sequence of end-joining junctions (see below) and amplification of multiple, related DNA fragments is prone to ‘template-switching’ artifacts and amplification bias. In addition, the cloned-survivor approach improves the chance of revealing repair junctions in unanticipated locations since clones that initially fail to reveal a junction can be specifically targeted for further analysis.Figure 2.

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