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Genetic Regulation of Dna2 Localization During the DNA Damage Response.

Yimit A, Riffle M, Brown GW - G3 (Bethesda) (2015)

Bottom Line: We find that Dna2-GFP focus formation occurs mainly during S phase in unperturbed cells.To systematically identify genes that affect Dna2 focus formation, we crossed Dna2-GFP into 4293 nonessential gene deletion mutants and assessed Dna2-GFP nuclear focus formation after phleomycin treatment.We identified 37 gene deletions that affect Dna2-GFP focus formation, 12 with fewer foci and 25 with increased foci.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.

No MeSH data available.


Related in: MedlinePlus

Identification of genes that affect Dna2-GFP focus formation. (A) The fraction of cells with Dna2-GFP foci in 12 gene deletions with fewer Dna2 foci after treatment with phleomycin and in 25 gene deletions with increased Dna2 foci either in untreated cells or after treatment with phleomycin is plotted for two replicates. (B) The 37 confirmed genes that affect Dna2 foci abundance are organized in a network with nodes colored according to function. Edges are in red for gene deletions with fewer foci and in green for gene deletions with increased foci. (C) Dna2-GFP fluorescence intensity after treatment with phleomycin is plotted for whole cells (dark blue; n = 10) and nuclei (light blue; n = 10). Horizontal bars indicate the median Dna2-GFP fluorescence in each compartment for each mutant. Horizontal lines mark the median Dna2-GFP fluorescence in each compartment for wild-type. (D) Dna2-GFP nuclear focus intensity after treatment with phleomycin is plotted for each mutant (n = 12 to 20) and wild-type. Horizontal bars indicate the median focus intensity for each mutant, and the horizontal line marks the median focus intensity for wild-type.
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fig4: Identification of genes that affect Dna2-GFP focus formation. (A) The fraction of cells with Dna2-GFP foci in 12 gene deletions with fewer Dna2 foci after treatment with phleomycin and in 25 gene deletions with increased Dna2 foci either in untreated cells or after treatment with phleomycin is plotted for two replicates. (B) The 37 confirmed genes that affect Dna2 foci abundance are organized in a network with nodes colored according to function. Edges are in red for gene deletions with fewer foci and in green for gene deletions with increased foci. (C) Dna2-GFP fluorescence intensity after treatment with phleomycin is plotted for whole cells (dark blue; n = 10) and nuclei (light blue; n = 10). Horizontal bars indicate the median Dna2-GFP fluorescence in each compartment for each mutant. Horizontal lines mark the median Dna2-GFP fluorescence in each compartment for wild-type. (D) Dna2-GFP nuclear focus intensity after treatment with phleomycin is plotted for each mutant (n = 12 to 20) and wild-type. Horizontal bars indicate the median focus intensity for each mutant, and the horizontal line marks the median focus intensity for wild-type.

Mentions: To systematically identify the genetic requirements for Dna2 focus formation, we screened a collection of 4293 haploid nonessential gene deletion mutants (Costanzo et al. 2010; Giaever et al. 2002) in the absence and presence of phleomycin. Dna2-GFP foci were visualized by high-throughput confocal microscopy and scored by visual inspection. All images from the screen are available from the Yeast Resource Center Public Image Repository (Riffle and Davis 2010) at http://images.yeastrc.org/yimit-2015. Forty-seven genes were identified that affected Dna2-GFP focus formation, either by increasing focus formation in untreated cells (32 genes) or by decreasing focus formation in phleomycin-treated cells (15 genes) (Table S4). These positives were reimaged in low throughput before and after treatment with phleomycin for 2 hr, and foci in the resulting images were quantified. We confirmed that 12 mutants showed a decrease (P < 0.05) in the fraction of cells with a Dna2-GFP focus following phleomycin treatment, relative to wild-type (Figure 4A), and that 25 mutants had an increased (P < 0.05) fraction of cells with Dna2 foci relative to wild-type (Figure 4A). We identified three classes of mutants with increased Dna2 foci: those with increased spontaneous foci only (11), those with increased spontaneous and increased phleomycin-induced foci (7), and those with increased phleomycin-induced foci only (7) (Figure 4A). There are likely additional mutants in the deletion collection that cause increased Dna2 foci in phleomycin only, because this class was not scored in our primary screen.


Genetic Regulation of Dna2 Localization During the DNA Damage Response.

Yimit A, Riffle M, Brown GW - G3 (Bethesda) (2015)

Identification of genes that affect Dna2-GFP focus formation. (A) The fraction of cells with Dna2-GFP foci in 12 gene deletions with fewer Dna2 foci after treatment with phleomycin and in 25 gene deletions with increased Dna2 foci either in untreated cells or after treatment with phleomycin is plotted for two replicates. (B) The 37 confirmed genes that affect Dna2 foci abundance are organized in a network with nodes colored according to function. Edges are in red for gene deletions with fewer foci and in green for gene deletions with increased foci. (C) Dna2-GFP fluorescence intensity after treatment with phleomycin is plotted for whole cells (dark blue; n = 10) and nuclei (light blue; n = 10). Horizontal bars indicate the median Dna2-GFP fluorescence in each compartment for each mutant. Horizontal lines mark the median Dna2-GFP fluorescence in each compartment for wild-type. (D) Dna2-GFP nuclear focus intensity after treatment with phleomycin is plotted for each mutant (n = 12 to 20) and wild-type. Horizontal bars indicate the median focus intensity for each mutant, and the horizontal line marks the median focus intensity for wild-type.
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fig4: Identification of genes that affect Dna2-GFP focus formation. (A) The fraction of cells with Dna2-GFP foci in 12 gene deletions with fewer Dna2 foci after treatment with phleomycin and in 25 gene deletions with increased Dna2 foci either in untreated cells or after treatment with phleomycin is plotted for two replicates. (B) The 37 confirmed genes that affect Dna2 foci abundance are organized in a network with nodes colored according to function. Edges are in red for gene deletions with fewer foci and in green for gene deletions with increased foci. (C) Dna2-GFP fluorescence intensity after treatment with phleomycin is plotted for whole cells (dark blue; n = 10) and nuclei (light blue; n = 10). Horizontal bars indicate the median Dna2-GFP fluorescence in each compartment for each mutant. Horizontal lines mark the median Dna2-GFP fluorescence in each compartment for wild-type. (D) Dna2-GFP nuclear focus intensity after treatment with phleomycin is plotted for each mutant (n = 12 to 20) and wild-type. Horizontal bars indicate the median focus intensity for each mutant, and the horizontal line marks the median focus intensity for wild-type.
Mentions: To systematically identify the genetic requirements for Dna2 focus formation, we screened a collection of 4293 haploid nonessential gene deletion mutants (Costanzo et al. 2010; Giaever et al. 2002) in the absence and presence of phleomycin. Dna2-GFP foci were visualized by high-throughput confocal microscopy and scored by visual inspection. All images from the screen are available from the Yeast Resource Center Public Image Repository (Riffle and Davis 2010) at http://images.yeastrc.org/yimit-2015. Forty-seven genes were identified that affected Dna2-GFP focus formation, either by increasing focus formation in untreated cells (32 genes) or by decreasing focus formation in phleomycin-treated cells (15 genes) (Table S4). These positives were reimaged in low throughput before and after treatment with phleomycin for 2 hr, and foci in the resulting images were quantified. We confirmed that 12 mutants showed a decrease (P < 0.05) in the fraction of cells with a Dna2-GFP focus following phleomycin treatment, relative to wild-type (Figure 4A), and that 25 mutants had an increased (P < 0.05) fraction of cells with Dna2 foci relative to wild-type (Figure 4A). We identified three classes of mutants with increased Dna2 foci: those with increased spontaneous foci only (11), those with increased spontaneous and increased phleomycin-induced foci (7), and those with increased phleomycin-induced foci only (7) (Figure 4A). There are likely additional mutants in the deletion collection that cause increased Dna2 foci in phleomycin only, because this class was not scored in our primary screen.

Bottom Line: We find that Dna2-GFP focus formation occurs mainly during S phase in unperturbed cells.To systematically identify genes that affect Dna2 focus formation, we crossed Dna2-GFP into 4293 nonessential gene deletion mutants and assessed Dna2-GFP nuclear focus formation after phleomycin treatment.We identified 37 gene deletions that affect Dna2-GFP focus formation, 12 with fewer foci and 25 with increased foci.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada.

No MeSH data available.


Related in: MedlinePlus