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Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes.

Glover L, McCulloch R, Horn D - Nucleic Acids Res. (2008)

Bottom Line: HR displayed a strong preference for the allelic template but also the capacity to interact with homologous sequence on heterologous chromosomes.Intra-chromosomal joining was predominantly, and possibly exclusively, microhomology mediated, a situation unique among organisms examined to date.These DSBR pathways available to T. brucei likely underlie patterns of antigenic variation and the evolution of the vast VSG gene family.

View Article: PubMed Central - PubMed

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

ABSTRACT
Genetic diversity in fungi and mammals is generated through mitotic double-strand break-repair (DSBR), typically involving homologous recombination (HR) or non-homologous end joining (NHEJ). Microhomology-mediated joining appears to serve a subsidiary function. The African trypanosome, a divergent protozoan parasite, relies upon rearrangement of subtelomeric variant surface glycoprotein (VSG) genes to achieve antigenic variation. Evidence suggests an absence of NHEJ but chromosomal repair remains largely unexplored. We used a system based on I-SceI meganuclease and monitored temporally constrained DSBR at a specific chromosomal site in bloodstream form Trypanosoma brucei. In response to the lesion, adjacent single-stranded DNA was generated; the homologous strand-exchange factor, Rad51, accumulated into foci; a G(2)M checkpoint was activated and >50% of cells displayed successful repair. Quantitative analysis of DSBR pathways employed indicated that inter-chromosomal HR dominated. HR displayed a strong preference for the allelic template but also the capacity to interact with homologous sequence on heterologous chromosomes. Intra-chromosomal joining was predominantly, and possibly exclusively, microhomology mediated, a situation unique among organisms examined to date. These DSBR pathways available to T. brucei likely underlie patterns of antigenic variation and the evolution of the vast VSG gene family.

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Rad51 accumulates at sub-nuclear foci in response to DSBs. (A) Immunofluorescence analysis of Rad51 in wild-type (WT) cells and in Sce2110 cells 9 h after I-SceI-induction. Rad51 signals are shown before and after deconvolution (d). DNA was counter-stained with DAPI. Scale bar, 5 μm. An expanded view of a nucleus with a prominent Rad51 focus is shown to the right. (B) Rad51 foci kinetics. The proportion of nuclei with Rad51 foci were counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Rad51 levels remain constant during DSBR. Western blotting with anti-Rad51 and a series of protein samples extracted at different times after I-SceI-induction. An equivalent Coomassie-stained gel served as a loading-control. The predicted Mwt of TbRad51 is ∼41 kDa.
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Figure 3: Rad51 accumulates at sub-nuclear foci in response to DSBs. (A) Immunofluorescence analysis of Rad51 in wild-type (WT) cells and in Sce2110 cells 9 h after I-SceI-induction. Rad51 signals are shown before and after deconvolution (d). DNA was counter-stained with DAPI. Scale bar, 5 μm. An expanded view of a nucleus with a prominent Rad51 focus is shown to the right. (B) Rad51 foci kinetics. The proportion of nuclei with Rad51 foci were counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Rad51 levels remain constant during DSBR. Western blotting with anti-Rad51 and a series of protein samples extracted at different times after I-SceI-induction. An equivalent Coomassie-stained gel served as a loading-control. The predicted Mwt of TbRad51 is ∼41 kDa.

Mentions: Rad51 (RecA in bacteria and RadA in archaebacteria) forms helical filaments on ssDNA and catalyses homologous strand exchange (35). Several recombination proteins, including Rad51, show diffuse localization in undamaged cells, but localize to sites of DNA damage forming sub-nuclear foci detectable by microscopy. Since we demonstrated the processing of single DSBs to generate ssDNA, we also wanted to determine whether these DSBs could trigger the assembly of Rad51 foci. We carried out immunofluorescence analysis using anti-Rad51 to compare wild-type cells to cells with I-SceI-induced lesions. Figure 3A shows that Rad51 was enriched in the nuclei of wild-type cells but the signal was typically diffuse (left-hand panels) in contrast to the situation in cells with a lesion, where a substantial proportion displayed Rad51 foci (right-hand panels). We used deconvolution to enhance foci and these images more clearly show nuclear foci in many induced cells and the absence of foci in the majority of wild-type cells. Consistent with previous work (29), foci were detected in only ∼1% of wild-type nuclei, likely reflecting the recruitment of repair proteins to sites of spontaneous DNA damage. We then counted Rad51 foci in cells induced for different periods of time (Figure 3B). In cells with a lesion on chromosome 11, there was a rapid increase in the proportion with nuclear foci, peaking at ∼30% 9 h after induction. A high proportion of cells with foci was still detected after 24 h but had diminished to background after 48 h (Figure 3B).Figure 3.


Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes.

Glover L, McCulloch R, Horn D - Nucleic Acids Res. (2008)

Rad51 accumulates at sub-nuclear foci in response to DSBs. (A) Immunofluorescence analysis of Rad51 in wild-type (WT) cells and in Sce2110 cells 9 h after I-SceI-induction. Rad51 signals are shown before and after deconvolution (d). DNA was counter-stained with DAPI. Scale bar, 5 μm. An expanded view of a nucleus with a prominent Rad51 focus is shown to the right. (B) Rad51 foci kinetics. The proportion of nuclei with Rad51 foci were counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Rad51 levels remain constant during DSBR. Western blotting with anti-Rad51 and a series of protein samples extracted at different times after I-SceI-induction. An equivalent Coomassie-stained gel served as a loading-control. The predicted Mwt of TbRad51 is ∼41 kDa.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Rad51 accumulates at sub-nuclear foci in response to DSBs. (A) Immunofluorescence analysis of Rad51 in wild-type (WT) cells and in Sce2110 cells 9 h after I-SceI-induction. Rad51 signals are shown before and after deconvolution (d). DNA was counter-stained with DAPI. Scale bar, 5 μm. An expanded view of a nucleus with a prominent Rad51 focus is shown to the right. (B) Rad51 foci kinetics. The proportion of nuclei with Rad51 foci were counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Rad51 levels remain constant during DSBR. Western blotting with anti-Rad51 and a series of protein samples extracted at different times after I-SceI-induction. An equivalent Coomassie-stained gel served as a loading-control. The predicted Mwt of TbRad51 is ∼41 kDa.
Mentions: Rad51 (RecA in bacteria and RadA in archaebacteria) forms helical filaments on ssDNA and catalyses homologous strand exchange (35). Several recombination proteins, including Rad51, show diffuse localization in undamaged cells, but localize to sites of DNA damage forming sub-nuclear foci detectable by microscopy. Since we demonstrated the processing of single DSBs to generate ssDNA, we also wanted to determine whether these DSBs could trigger the assembly of Rad51 foci. We carried out immunofluorescence analysis using anti-Rad51 to compare wild-type cells to cells with I-SceI-induced lesions. Figure 3A shows that Rad51 was enriched in the nuclei of wild-type cells but the signal was typically diffuse (left-hand panels) in contrast to the situation in cells with a lesion, where a substantial proportion displayed Rad51 foci (right-hand panels). We used deconvolution to enhance foci and these images more clearly show nuclear foci in many induced cells and the absence of foci in the majority of wild-type cells. Consistent with previous work (29), foci were detected in only ∼1% of wild-type nuclei, likely reflecting the recruitment of repair proteins to sites of spontaneous DNA damage. We then counted Rad51 foci in cells induced for different periods of time (Figure 3B). In cells with a lesion on chromosome 11, there was a rapid increase in the proportion with nuclear foci, peaking at ∼30% 9 h after induction. A high proportion of cells with foci was still detected after 24 h but had diminished to background after 48 h (Figure 3B).Figure 3.

Bottom Line: HR displayed a strong preference for the allelic template but also the capacity to interact with homologous sequence on heterologous chromosomes.Intra-chromosomal joining was predominantly, and possibly exclusively, microhomology mediated, a situation unique among organisms examined to date.These DSBR pathways available to T. brucei likely underlie patterns of antigenic variation and the evolution of the vast VSG gene family.

View Article: PubMed Central - PubMed

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

ABSTRACT
Genetic diversity in fungi and mammals is generated through mitotic double-strand break-repair (DSBR), typically involving homologous recombination (HR) or non-homologous end joining (NHEJ). Microhomology-mediated joining appears to serve a subsidiary function. The African trypanosome, a divergent protozoan parasite, relies upon rearrangement of subtelomeric variant surface glycoprotein (VSG) genes to achieve antigenic variation. Evidence suggests an absence of NHEJ but chromosomal repair remains largely unexplored. We used a system based on I-SceI meganuclease and monitored temporally constrained DSBR at a specific chromosomal site in bloodstream form Trypanosoma brucei. In response to the lesion, adjacent single-stranded DNA was generated; the homologous strand-exchange factor, Rad51, accumulated into foci; a G(2)M checkpoint was activated and >50% of cells displayed successful repair. Quantitative analysis of DSBR pathways employed indicated that inter-chromosomal HR dominated. HR displayed a strong preference for the allelic template but also the capacity to interact with homologous sequence on heterologous chromosomes. Intra-chromosomal joining was predominantly, and possibly exclusively, microhomology mediated, a situation unique among organisms examined to date. These DSBR pathways available to T. brucei likely underlie patterns of antigenic variation and the evolution of the vast VSG gene family.

Show MeSH
Related in: MedlinePlus