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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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An experimental system to study end-joining. (A) The schematic maps illustrate the Tb11.02.2110 alleles in wild-type (WT), RsPa 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 RsPa controls. The RΔ and RsPa 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 RsPa 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 RsPa/Δ2110b survivors. The TUB sequences flanking the RsP cassette promote RsP pre-mRNA trans-splicing and polyadenylation and also allow ectopic HR which replaces RsP with an αTUB gene copied from chromosome 1.
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Figure 1: An experimental system to study end-joining. (A) The schematic maps illustrate the Tb11.02.2110 alleles in wild-type (WT), RsPa 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 RsPa controls. The RΔ and RsPa 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 RsPa 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 RsPa/Δ2110b survivors. The TUB sequences flanking the RsP cassette promote RsP pre-mRNA trans-splicing and polyadenylation and also allow ectopic HR which replaces RsP with an αTUB gene copied from chromosome 1.

Mentions: We previously used the I-SceI meganuclease to introduce a single DSB on T. brucei chromosome 11a (6); the RsPa strain used contains a tetracycline-inducible I-SceI gene and a single I-SceI cleavage site adjacent to the Tb11.02.2110 gene (Figure 1A). Using this strain, >50% of cells survived I-SceI induction and repaired the break and ∼85% of these survivors underwent allelic HR using the 2110b-allele as a repair template. In addition, two alternatives to allelic HR were revealed; among 26-independent repair events, three clones displayed ectopic HR and two displayed MMEJ (6), an insufficient number for any detailed analysis. To facilitate the isolation of survivors that display end joining and to characterize MMEJ in a chromosomal context, we devised an experimental system to eliminate survivors that use the major repair pathway of allelic HR. This was achieved by replacing the 2110b-allele and other break-adjacent homology with a NEO selectable marker (Figure 1A). In the resulting RsPa/Δ2110b (RΔ) strains, allelic HR was expected to cause loss of heterozygocity, loss of the remaining 2110a allele and, because the encoded N-terminal protein acetyltransferase is essential for growth (16), cell death. Disruption of a single 2110 allele had no detectable impact on the growth rate of the RΔ strains (data not shown).Figure 1.


Microhomology-mediated deletion and gene conversion in African trypanosomes.

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

An experimental system to study end-joining. (A) The schematic maps illustrate the Tb11.02.2110 alleles in wild-type (WT), RsPa 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 RsPa controls. The RΔ and RsPa 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 RsPa 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 RsPa/Δ2110b survivors. The TUB sequences flanking the RsP cassette promote RsP pre-mRNA trans-splicing and polyadenylation and also allow ectopic HR which replaces RsP with an αTUB gene copied from chromosome 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: An experimental system to study end-joining. (A) The schematic maps illustrate the Tb11.02.2110 alleles in wild-type (WT), RsPa 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 RsPa controls. The RΔ and RsPa 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 RsPa 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 RsPa/Δ2110b survivors. The TUB sequences flanking the RsP cassette promote RsP pre-mRNA trans-splicing and polyadenylation and also allow ectopic HR which replaces RsP with an αTUB gene copied from chromosome 1.
Mentions: We previously used the I-SceI meganuclease to introduce a single DSB on T. brucei chromosome 11a (6); the RsPa strain used contains a tetracycline-inducible I-SceI gene and a single I-SceI cleavage site adjacent to the Tb11.02.2110 gene (Figure 1A). Using this strain, >50% of cells survived I-SceI induction and repaired the break and ∼85% of these survivors underwent allelic HR using the 2110b-allele as a repair template. In addition, two alternatives to allelic HR were revealed; among 26-independent repair events, three clones displayed ectopic HR and two displayed MMEJ (6), an insufficient number for any detailed analysis. To facilitate the isolation of survivors that display end joining and to characterize MMEJ in a chromosomal context, we devised an experimental system to eliminate survivors that use the major repair pathway of allelic HR. This was achieved by replacing the 2110b-allele and other break-adjacent homology with a NEO selectable marker (Figure 1A). In the resulting RsPa/Δ2110b (RΔ) strains, allelic HR was expected to cause loss of heterozygocity, loss of the remaining 2110a allele and, because the encoded N-terminal protein acetyltransferase is essential for growth (16), cell death. Disruption of a single 2110 allele had no detectable impact on the growth rate of the RΔ strains (data not shown).Figure 1.

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