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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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One-sided MMEJ-based gene conversion is RAD51 dependent. (A) PCR assays indicate a similar pattern of MMEJ-based deletion in RAD51 and rad51  strains (upper panel) while one-sided MMEJ-based gene conversion is ablated in rad51  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.
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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 strains (upper panel) while one-sided MMEJ-based gene conversion is ablated in rad51 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.

Mentions: MMEJ is typically RAD51-independent. To assess the role of RAD51 in chromosomal MMEJ-based deletion and gene conversion, we applied a PCR assay to populations of survivors from RsPa strains with wild-type RAD51 expression or with rad51 disrupted. An assay for MMEJ-based deletions indicated robust activity in the absence of RAD51 (Figure 6A, upper panel), and sequencing of nine rad51 survivors revealed exclusively MMEJ-based deletions (data not shown). In contrast, one-sided MMEJ-based gene conversion was specifically ablated in the rad51 strain (Figure 6A, lower panel). Sequencing confirmed that the major products detected using this assay in wild-type RAD51 cells both represented one-sided MMEJ (Figure 6B and C). Thus, one-sided MMEJ-based gene conversion can use allelic or ectopic homology on chromosome 1. The RAD51-requirement suggests that gene conversion is initiated by HR within tubulin sequence and resolved by MMEJ. We also analyzed an RsPa survivor for which we previously failed to identify a repair mechanism [see Figure 5B, lane 4 in (6)] and this survivor was also found to have arisen through one-sided ectopic MMEJ (Figure 6B and C, iii). Segments copied from chromosome 1 in these RAD51-dependent, one-sided MMEJ reactions ranged from 28 to 1084 bp.Figure 6.


Microhomology-mediated deletion and gene conversion in African trypanosomes.

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

One-sided MMEJ-based gene conversion is RAD51 dependent. (A) PCR assays indicate a similar pattern of MMEJ-based deletion in RAD51 and rad51  strains (upper panel) while one-sided MMEJ-based gene conversion is ablated in rad51  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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045614&req=5

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 strains (upper panel) while one-sided MMEJ-based gene conversion is ablated in rad51 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.
Mentions: MMEJ is typically RAD51-independent. To assess the role of RAD51 in chromosomal MMEJ-based deletion and gene conversion, we applied a PCR assay to populations of survivors from RsPa strains with wild-type RAD51 expression or with rad51 disrupted. An assay for MMEJ-based deletions indicated robust activity in the absence of RAD51 (Figure 6A, upper panel), and sequencing of nine rad51 survivors revealed exclusively MMEJ-based deletions (data not shown). In contrast, one-sided MMEJ-based gene conversion was specifically ablated in the rad51 strain (Figure 6A, lower panel). Sequencing confirmed that the major products detected using this assay in wild-type RAD51 cells both represented one-sided MMEJ (Figure 6B and C). Thus, one-sided MMEJ-based gene conversion can use allelic or ectopic homology on chromosome 1. The RAD51-requirement suggests that gene conversion is initiated by HR within tubulin sequence and resolved by MMEJ. We also analyzed an RsPa survivor for which we previously failed to identify a repair mechanism [see Figure 5B, lane 4 in (6)] and this survivor was also found to have arisen through one-sided ectopic MMEJ (Figure 6B and C, iii). Segments copied from chromosome 1 in these RAD51-dependent, one-sided MMEJ reactions ranged from 28 to 1084 bp.Figure 6.

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