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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

Dna2-GFP focus formation in G1 and asynchronous cells. (A) Logarithmic phase asynchronous cells or cells arrested in G1 were exposed to phleomycin (5 μg/ml) and imaged by confocal microscopy to detect Dna2-GFP foci. (B) The number of Dna2-GFP foci per cell was quantified for the G1-arrested and asynchronous cells. At least 100 cells were analyzed in three independent experiments, and the percent of cells with at least one Dna2-GFP focus is plotted for each.
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fig1: Dna2-GFP focus formation in G1 and asynchronous cells. (A) Logarithmic phase asynchronous cells or cells arrested in G1 were exposed to phleomycin (5 μg/ml) and imaged by confocal microscopy to detect Dna2-GFP foci. (B) The number of Dna2-GFP foci per cell was quantified for the G1-arrested and asynchronous cells. At least 100 cells were analyzed in three independent experiments, and the percent of cells with at least one Dna2-GFP focus is plotted for each.

Mentions: Dna2, like many DNA damage response proteins, forms nuclear foci in response to double-strand breaks and DNA replication stress (Lisby and Rothstein 2009; Makhnevych et al. 2009; Tkach et al. 2012; Chen et al. 2011). In addition to being regulated by DNA damage, the intracellular localization of Dna2 is connected to cell cycle phase via CDK phosphorylation. In G1 arrested cells Dna2 is mainly cytoplasmic, whereas during S, G2, and M phases Dna2 displays a nuclear localization (Kosugi et al. 2009). To investigate the cell cycle distribution of Dna2 foci in unperturbed cells and cells with double-strand DNA breaks, we quantified Dna2-GFP foci in unbudded (G1), small budded (S), and large budded (G2) cells in both asynchronous cultures and cultures treated with phleomycin. Phleomycin, an antibiotic of the bleomycin family, causes free radical–mediated DNA damage, including double-strand breaks (Moore 1988; Sleigh 1976). In unperturbed cells, Dna2 foci were mainly found in S phase in 24% of small budded cells (Table 1), suggesting that Dna2 foci can arise during DNA replication. Following 2 hr of treatment with phleomycin, Dna2 foci were found in small budded and large budded cells, but rarely in unbudded (G1) cells (Table 1). We arrested cells in G1 with mating pheromone and treated the arrested cells with phleomycin (Figure 1, A and B), confirming that Dna2 foci do not form efficiently during G1 phase. These results are in agreement with the established roles of Dna2 in Okazaki fragment maturation (in S phase) and roles in double-strand break repair (DNA resection during G2/M phase).


Genetic Regulation of Dna2 Localization During the DNA Damage Response.

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

Dna2-GFP focus formation in G1 and asynchronous cells. (A) Logarithmic phase asynchronous cells or cells arrested in G1 were exposed to phleomycin (5 μg/ml) and imaged by confocal microscopy to detect Dna2-GFP foci. (B) The number of Dna2-GFP foci per cell was quantified for the G1-arrested and asynchronous cells. At least 100 cells were analyzed in three independent experiments, and the percent of cells with at least one Dna2-GFP focus is plotted for each.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555230&req=5

fig1: Dna2-GFP focus formation in G1 and asynchronous cells. (A) Logarithmic phase asynchronous cells or cells arrested in G1 were exposed to phleomycin (5 μg/ml) and imaged by confocal microscopy to detect Dna2-GFP foci. (B) The number of Dna2-GFP foci per cell was quantified for the G1-arrested and asynchronous cells. At least 100 cells were analyzed in three independent experiments, and the percent of cells with at least one Dna2-GFP focus is plotted for each.
Mentions: Dna2, like many DNA damage response proteins, forms nuclear foci in response to double-strand breaks and DNA replication stress (Lisby and Rothstein 2009; Makhnevych et al. 2009; Tkach et al. 2012; Chen et al. 2011). In addition to being regulated by DNA damage, the intracellular localization of Dna2 is connected to cell cycle phase via CDK phosphorylation. In G1 arrested cells Dna2 is mainly cytoplasmic, whereas during S, G2, and M phases Dna2 displays a nuclear localization (Kosugi et al. 2009). To investigate the cell cycle distribution of Dna2 foci in unperturbed cells and cells with double-strand DNA breaks, we quantified Dna2-GFP foci in unbudded (G1), small budded (S), and large budded (G2) cells in both asynchronous cultures and cultures treated with phleomycin. Phleomycin, an antibiotic of the bleomycin family, causes free radical–mediated DNA damage, including double-strand breaks (Moore 1988; Sleigh 1976). In unperturbed cells, Dna2 foci were mainly found in S phase in 24% of small budded cells (Table 1), suggesting that Dna2 foci can arise during DNA replication. Following 2 hr of treatment with phleomycin, Dna2 foci were found in small budded and large budded cells, but rarely in unbudded (G1) cells (Table 1). We arrested cells in G1 with mating pheromone and treated the arrested cells with phleomycin (Figure 1, A and B), confirming that Dna2 foci do not form efficiently during G1 phase. These results are in agreement with the established roles of Dna2 in Okazaki fragment maturation (in S phase) and roles in double-strand break repair (DNA resection during G2/M phase).

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