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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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Physical monitoring of DNA resection and repair. (A) Monitoring ssDNA adjacent to the lesion by slot-blot analysis. Genomic DNA samples were extracted at various times following I-SceI-induction. Ninety percent of each sample was ‘native’ (n) and the remainder denatured (d). The probes used on each blot are indicated on the right. 7240 is a distal, chromosome 11 control. The schematic map indicates the location of the probes (black) in relation to the lesion (DSB). (B) Kinetics of ssDNA formation. Phoshorimager analysis was used to quantify the signals in (A). (C) Monitoring repair by Southern blot analysis. Genomic DNA extracted at various times following I-SceI-induction was digested with HindIII and subjected to Southern blot analysis using the probes indicated. Arrowheads indicate the fragments expected following HR between chromosome 11a and 11b or the TUB locus on chromosome 1 (see Figure 1). For chromosome 11, 7240 served as a loading control. The schematic illustrates dominant allelic HR with chromosome 11b. (D) Kinetics of repair by HR with chromosome 11b. Phoshorimager analysis was used to quantify the signals in (C).
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Figure 2: Physical monitoring of DNA resection and repair. (A) Monitoring ssDNA adjacent to the lesion by slot-blot analysis. Genomic DNA samples were extracted at various times following I-SceI-induction. Ninety percent of each sample was ‘native’ (n) and the remainder denatured (d). The probes used on each blot are indicated on the right. 7240 is a distal, chromosome 11 control. The schematic map indicates the location of the probes (black) in relation to the lesion (DSB). (B) Kinetics of ssDNA formation. Phoshorimager analysis was used to quantify the signals in (A). (C) Monitoring repair by Southern blot analysis. Genomic DNA extracted at various times following I-SceI-induction was digested with HindIII and subjected to Southern blot analysis using the probes indicated. Arrowheads indicate the fragments expected following HR between chromosome 11a and 11b or the TUB locus on chromosome 1 (see Figure 1). For chromosome 11, 7240 served as a loading control. The schematic illustrates dominant allelic HR with chromosome 11b. (D) Kinetics of repair by HR with chromosome 11b. Phoshorimager analysis was used to quantify the signals in (C).

Mentions: Introduction of a DSB on T. brucei chromosome 11 was controlled by placing the I-SceI gene downstream of a Tet-inducible promoter. Upon addition of Tet to the medium, the enzyme generated a DSB at the RSP locus. To monitor the kinetics of repair, we extracted DNA from cells at different time points following I-SceI-induction. Prior to initiation of mitotic HR, a DSB must be processed by degradation of the 5' strand in a process known as resection to generate single-stranded DNA (ssDNA) with a 3′ end. The ssDNA is thought to generate the signal for the DNA damage checkpoint, and is the substrate for Rad51 binding to initiate a search for a suitable homologous repair template (7). To physically monitor ssDNA adjacent to the lesion on chromosome 11, hybridization analysis was applied to native chromosomal DNA samples (Figure 2A and B); hybridization to native DNA will occur only if ssDNA is present. Denatured samples were analysed in parallel to control for loading. To increase sensitivity, we loaded nine times more native DNA relative to denatured DNA for each time point.Figure 2.


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

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

Physical monitoring of DNA resection and repair. (A) Monitoring ssDNA adjacent to the lesion by slot-blot analysis. Genomic DNA samples were extracted at various times following I-SceI-induction. Ninety percent of each sample was ‘native’ (n) and the remainder denatured (d). The probes used on each blot are indicated on the right. 7240 is a distal, chromosome 11 control. The schematic map indicates the location of the probes (black) in relation to the lesion (DSB). (B) Kinetics of ssDNA formation. Phoshorimager analysis was used to quantify the signals in (A). (C) Monitoring repair by Southern blot analysis. Genomic DNA extracted at various times following I-SceI-induction was digested with HindIII and subjected to Southern blot analysis using the probes indicated. Arrowheads indicate the fragments expected following HR between chromosome 11a and 11b or the TUB locus on chromosome 1 (see Figure 1). For chromosome 11, 7240 served as a loading control. The schematic illustrates dominant allelic HR with chromosome 11b. (D) Kinetics of repair by HR with chromosome 11b. Phoshorimager analysis was used to quantify the signals in (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2377438&req=5

Figure 2: Physical monitoring of DNA resection and repair. (A) Monitoring ssDNA adjacent to the lesion by slot-blot analysis. Genomic DNA samples were extracted at various times following I-SceI-induction. Ninety percent of each sample was ‘native’ (n) and the remainder denatured (d). The probes used on each blot are indicated on the right. 7240 is a distal, chromosome 11 control. The schematic map indicates the location of the probes (black) in relation to the lesion (DSB). (B) Kinetics of ssDNA formation. Phoshorimager analysis was used to quantify the signals in (A). (C) Monitoring repair by Southern blot analysis. Genomic DNA extracted at various times following I-SceI-induction was digested with HindIII and subjected to Southern blot analysis using the probes indicated. Arrowheads indicate the fragments expected following HR between chromosome 11a and 11b or the TUB locus on chromosome 1 (see Figure 1). For chromosome 11, 7240 served as a loading control. The schematic illustrates dominant allelic HR with chromosome 11b. (D) Kinetics of repair by HR with chromosome 11b. Phoshorimager analysis was used to quantify the signals in (C).
Mentions: Introduction of a DSB on T. brucei chromosome 11 was controlled by placing the I-SceI gene downstream of a Tet-inducible promoter. Upon addition of Tet to the medium, the enzyme generated a DSB at the RSP locus. To monitor the kinetics of repair, we extracted DNA from cells at different time points following I-SceI-induction. Prior to initiation of mitotic HR, a DSB must be processed by degradation of the 5' strand in a process known as resection to generate single-stranded DNA (ssDNA) with a 3′ end. The ssDNA is thought to generate the signal for the DNA damage checkpoint, and is the substrate for Rad51 binding to initiate a search for a suitable homologous repair template (7). To physically monitor ssDNA adjacent to the lesion on chromosome 11, hybridization analysis was applied to native chromosomal DNA samples (Figure 2A and B); hybridization to native DNA will occur only if ssDNA is present. Denatured samples were analysed in parallel to control for loading. To increase sensitivity, we loaded nine times more native DNA relative to denatured DNA for each time point.Figure 2.

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