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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 foci and the G2M cell cycle checkpoint. Cells were processed for Rad51 immunofluorescence microscopy and DNA was counter-stained with DAPI. (A) The bar-chart shows the proportion of Sce2110 cells at different phases of the cell cycle with zero, one or two Rad51 foci 12 h after I-SceI-induction. Cell cycle phase was defined by the number of nuclei (N) and kinetoplasts (K) as determined by DAPI staining. n = 50 at each cell cycle phase. Error bars, SD. (B) G2M phase (1N2K) kinetics. The proportion of 1N2K cells was counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Immunofluorescence analysis of Rad51 in Sce2110 cells after I-SceI-induction. Rad51 signals are shown after deconvolution (d). N, nucleus; K, kinetoplast. Scale bar, 5 μm.
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Figure 4: Rad51 foci and the G2M cell cycle checkpoint. Cells were processed for Rad51 immunofluorescence microscopy and DNA was counter-stained with DAPI. (A) The bar-chart shows the proportion of Sce2110 cells at different phases of the cell cycle with zero, one or two Rad51 foci 12 h after I-SceI-induction. Cell cycle phase was defined by the number of nuclei (N) and kinetoplasts (K) as determined by DAPI staining. n = 50 at each cell cycle phase. Error bars, SD. (B) G2M phase (1N2K) kinetics. The proportion of 1N2K cells was counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Immunofluorescence analysis of Rad51 in Sce2110 cells after I-SceI-induction. Rad51 signals are shown after deconvolution (d). N, nucleus; K, kinetoplast. Scale bar, 5 μm.

Mentions: A single DNA-DSB can trigger a DNA damage checkpoint that arrests the cell cycle and allows time for repair prior to further progression thereby suppressing deleterious genome rearrangements (7). Efficient and temporally constrained introduction of DSBs in T. brucei provided an opportunity to explore DNA damage checkpoint control. No method is available to synchronize bloodstream form T. brucei, but nuclear and mitochondrial (kinetoplast) DNA, stained with DAPI, provide excellent cytological markers that define position in the cell cycle (38). In bloodstream form cells, ∼80% of cells display a single nucleus and a single kinetoplast (1N1K) indicating earlier phases of the cell cycle (G1/S). A single nucleus and two kinetoplasts (1N2K) indicate late nuclear G2 and two nuclei and two kinetoplasts (2N2K) indicate completion of mitosis. First, we examined the proportion of cells with Rad51 foci in these three categories after 12 h of I-SceI induction. We had also noted that some cells had two Rad51 foci, so we subdivided each category into cells with zero, one or two foci (Figure 4A).Figure 4.


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

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

Rad51 foci and the G2M cell cycle checkpoint. Cells were processed for Rad51 immunofluorescence microscopy and DNA was counter-stained with DAPI. (A) The bar-chart shows the proportion of Sce2110 cells at different phases of the cell cycle with zero, one or two Rad51 foci 12 h after I-SceI-induction. Cell cycle phase was defined by the number of nuclei (N) and kinetoplasts (K) as determined by DAPI staining. n = 50 at each cell cycle phase. Error bars, SD. (B) G2M phase (1N2K) kinetics. The proportion of 1N2K cells was counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Immunofluorescence analysis of Rad51 in Sce2110 cells after I-SceI-induction. Rad51 signals are shown after deconvolution (d). N, nucleus; K, kinetoplast. Scale bar, 5 μm.
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Figure 4: Rad51 foci and the G2M cell cycle checkpoint. Cells were processed for Rad51 immunofluorescence microscopy and DNA was counter-stained with DAPI. (A) The bar-chart shows the proportion of Sce2110 cells at different phases of the cell cycle with zero, one or two Rad51 foci 12 h after I-SceI-induction. Cell cycle phase was defined by the number of nuclei (N) and kinetoplasts (K) as determined by DAPI staining. n = 50 at each cell cycle phase. Error bars, SD. (B) G2M phase (1N2K) kinetics. The proportion of 1N2K cells was counted at different times after I-SceI-induction. n = 200 at each time point. Error bars, SD. (C) Immunofluorescence analysis of Rad51 in Sce2110 cells after I-SceI-induction. Rad51 signals are shown after deconvolution (d). N, nucleus; K, kinetoplast. Scale bar, 5 μm.
Mentions: A single DNA-DSB can trigger a DNA damage checkpoint that arrests the cell cycle and allows time for repair prior to further progression thereby suppressing deleterious genome rearrangements (7). Efficient and temporally constrained introduction of DSBs in T. brucei provided an opportunity to explore DNA damage checkpoint control. No method is available to synchronize bloodstream form T. brucei, but nuclear and mitochondrial (kinetoplast) DNA, stained with DAPI, provide excellent cytological markers that define position in the cell cycle (38). In bloodstream form cells, ∼80% of cells display a single nucleus and a single kinetoplast (1N1K) indicating earlier phases of the cell cycle (G1/S). A single nucleus and two kinetoplasts (1N2K) indicate late nuclear G2 and two nuclei and two kinetoplasts (2N2K) indicate completion of mitosis. First, we examined the proportion of cells with Rad51 foci in these three categories after 12 h of I-SceI induction. We had also noted that some cells had two Rad51 foci, so we subdivided each category into cells with zero, one or two foci (Figure 4A).Figure 4.

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