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Locus-specific control of DNA resection and suppression of subtelomeric VSG recombination by HAT3 in the African trypanosome.

Glover L, Horn D - Nucleic Acids Res. (2014)

Bottom Line: Although HAT3 promoted chromosome-internal recombination, it suppressed subtelomeric VSG recombination, and these locus-specific effects were mediated through differential production of ssDNA by DNA resection; HAT3 promoted chromosome-internal resection but suppressed subtelomeric resection.HAT3 also promoted resection at a second chromosome-internal locus comprising tandem-duplicated genes.HAT3 promotes ssDNA formation and recombination at chromosome-internal sites but has the opposite effect at a subtelomeric VSG.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

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Locus-dependent control of DNA resection by HAT3. Accumulation of ssDNA adjacent to a DSB was monitored by slot-blot analysis in the INT (A) and TEL (C) strains. Genomic DNA was extracted at the times indicated following I-SceI induction. Ninety percent of each sample was ‘native’ (n; where a hybridization signal with native DNA indicates the presence of ssDNA) and the remainder was denatured (d). The probes used on each blot are indicated on the right (see the schematic maps in Figures 1A and 4A). ssDNA signals from the INT (B) and TEL (D) strains were quantified by phosphorimager analysis as previously described (10).
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Figure 5: Locus-dependent control of DNA resection by HAT3. Accumulation of ssDNA adjacent to a DSB was monitored by slot-blot analysis in the INT (A) and TEL (C) strains. Genomic DNA was extracted at the times indicated following I-SceI induction. Ninety percent of each sample was ‘native’ (n; where a hybridization signal with native DNA indicates the presence of ssDNA) and the remainder was denatured (d). The probes used on each blot are indicated on the right (see the schematic maps in Figures 1A and 4A). ssDNA signals from the INT (B) and TEL (D) strains were quantified by phosphorimager analysis as previously described (10).

Mentions: We next sought the mechanism by which HAT3 and SIR2rp1 differentially control DNA repair at chromosome-internal and subtelomeric loci. Since DNA resection to generate ssDNA is required for RAD51-focus assembly at the site of the break, we monitored the kinetics of ssDNA accumulation over time adjacent to breaks in the INT and TEL strains (Figure 5). In INT cells, ssDNA accumulates adjacent to a break at the chromosome-internal locus 12 h after induction and declines thereafter (Figure 5A and B), as also described previously (10). In contrast, and consistent with the defects in RAD51-focal assembly and disassembly (Figure 2B and C), ssDNA fails to accumulate in INThat3 cells, and persists in INTsir2rp1 cells (Figure 5A and B).


Locus-specific control of DNA resection and suppression of subtelomeric VSG recombination by HAT3 in the African trypanosome.

Glover L, Horn D - Nucleic Acids Res. (2014)

Locus-dependent control of DNA resection by HAT3. Accumulation of ssDNA adjacent to a DSB was monitored by slot-blot analysis in the INT (A) and TEL (C) strains. Genomic DNA was extracted at the times indicated following I-SceI induction. Ninety percent of each sample was ‘native’ (n; where a hybridization signal with native DNA indicates the presence of ssDNA) and the remainder was denatured (d). The probes used on each blot are indicated on the right (see the schematic maps in Figures 1A and 4A). ssDNA signals from the INT (B) and TEL (D) strains were quantified by phosphorimager analysis as previously described (10).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227765&req=5

Figure 5: Locus-dependent control of DNA resection by HAT3. Accumulation of ssDNA adjacent to a DSB was monitored by slot-blot analysis in the INT (A) and TEL (C) strains. Genomic DNA was extracted at the times indicated following I-SceI induction. Ninety percent of each sample was ‘native’ (n; where a hybridization signal with native DNA indicates the presence of ssDNA) and the remainder was denatured (d). The probes used on each blot are indicated on the right (see the schematic maps in Figures 1A and 4A). ssDNA signals from the INT (B) and TEL (D) strains were quantified by phosphorimager analysis as previously described (10).
Mentions: We next sought the mechanism by which HAT3 and SIR2rp1 differentially control DNA repair at chromosome-internal and subtelomeric loci. Since DNA resection to generate ssDNA is required for RAD51-focus assembly at the site of the break, we monitored the kinetics of ssDNA accumulation over time adjacent to breaks in the INT and TEL strains (Figure 5). In INT cells, ssDNA accumulates adjacent to a break at the chromosome-internal locus 12 h after induction and declines thereafter (Figure 5A and B), as also described previously (10). In contrast, and consistent with the defects in RAD51-focal assembly and disassembly (Figure 2B and C), ssDNA fails to accumulate in INThat3 cells, and persists in INTsir2rp1 cells (Figure 5A and B).

Bottom Line: Although HAT3 promoted chromosome-internal recombination, it suppressed subtelomeric VSG recombination, and these locus-specific effects were mediated through differential production of ssDNA by DNA resection; HAT3 promoted chromosome-internal resection but suppressed subtelomeric resection.HAT3 also promoted resection at a second chromosome-internal locus comprising tandem-duplicated genes.HAT3 promotes ssDNA formation and recombination at chromosome-internal sites but has the opposite effect at a subtelomeric VSG.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

Show MeSH
Related in: MedlinePlus