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Fission yeast Rad26 responds to DNA damage independently of Rad3.

Wolkow TD, Enoch T - BMC Genet. (2003)

Bottom Line: We have discovered three distinct Rad26-GFP cellular structures.Formation of these structures did not require other checkpoint proteins.These data demonstrate that Rad26 can respond to genotoxic insult in the absence of Rad3 and the other checkpoint Rad proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. wolkow@rascal.med.harvard.edu

ABSTRACT

Background: The Rad26/Rad3 complex in fission yeast detects genotoxic insults and initiates the cell cycle arrest and recovery activities of the DNA damage checkpoint. To investigate how the Rad26/Rad3 complex performs these functions, we constructed and characterized Rad26-GFP.

Results: Rad26-GFP localized to approximately six nuclear dots in cycling cells. Following treatment with a DNA damaging agent, Rad26-GFP localization changed. Damaged cells contained one or two bright Rad26-GFP spots, in addition to smaller, more numerous Rad26-GFP speckles. Genetic analyses demonstrated that these Rad26-GFP patterns (dots, spots and speckles) were unaffected by mutations in other DNA damage checkpoint genes, including rad3+. Data obtained with our Rad26.T12-GFP fusion protein correlate spots with cell cycle arrest activities and speckles with DNA repair activities. In addition, physiological experiments demonstrated that rad26Delta and rad3Delta alleles confer sensitivity to a microtubule-depolymerizing drug.

Conclusion: We have discovered three distinct Rad26-GFP cellular structures. Formation of these structures did not require other checkpoint proteins. These data demonstrate that Rad26 can respond to genotoxic insult in the absence of Rad3 and the other checkpoint Rad proteins.

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Cycling cells contain Rad26-GFP dots. Cultures were grown at 30°C in liquid, complete media to O.D. 0.5 before cell wall digestion, Triton X-100 extraction and methanol fixation (see Methods). A. rad26+ (TE696) B. rad26-GFP (TE1197) C. rad9Δrad26-GFP (TE1193) Bar = 5 μm
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Figure 1: Cycling cells contain Rad26-GFP dots. Cultures were grown at 30°C in liquid, complete media to O.D. 0.5 before cell wall digestion, Triton X-100 extraction and methanol fixation (see Methods). A. rad26+ (TE696) B. rad26-GFP (TE1197) C. rad9Δrad26-GFP (TE1193) Bar = 5 μm

Mentions: Since the background fluorescence in live, cycling cells was too high to derive conclusions about Rad26-GFP localization (data not shown), we prepared cells using a technique previously described in a study of fission yeast chromatin binding proteins [42]. An asynchronous population of cycling cells was first permeabilized using Zymolyase (Seikagaku Corp., Tokyo), then extracted with 1% Triton X-100 and finally fixed with methanol. We did not observe GFP-fluorescence in negative-control, rad26+ cells (Figure 1A). However, rad26-GFP cells contained four to eight nuclear GFP dots (six on average, N = 100; Figure 1B). Non-extracted rad26-GFP cells, prepared in an otherwise identical manner, retained this GFP-fluorescence pattern (data not shown). We also obtained similar results using paraformaldehyde fixation, however in this case non-specific cytoplasmic background fluorescence was higher (data not shown).


Fission yeast Rad26 responds to DNA damage independently of Rad3.

Wolkow TD, Enoch T - BMC Genet. (2003)

Cycling cells contain Rad26-GFP dots. Cultures were grown at 30°C in liquid, complete media to O.D. 0.5 before cell wall digestion, Triton X-100 extraction and methanol fixation (see Methods). A. rad26+ (TE696) B. rad26-GFP (TE1197) C. rad9Δrad26-GFP (TE1193) Bar = 5 μm
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC155545&req=5

Figure 1: Cycling cells contain Rad26-GFP dots. Cultures were grown at 30°C in liquid, complete media to O.D. 0.5 before cell wall digestion, Triton X-100 extraction and methanol fixation (see Methods). A. rad26+ (TE696) B. rad26-GFP (TE1197) C. rad9Δrad26-GFP (TE1193) Bar = 5 μm
Mentions: Since the background fluorescence in live, cycling cells was too high to derive conclusions about Rad26-GFP localization (data not shown), we prepared cells using a technique previously described in a study of fission yeast chromatin binding proteins [42]. An asynchronous population of cycling cells was first permeabilized using Zymolyase (Seikagaku Corp., Tokyo), then extracted with 1% Triton X-100 and finally fixed with methanol. We did not observe GFP-fluorescence in negative-control, rad26+ cells (Figure 1A). However, rad26-GFP cells contained four to eight nuclear GFP dots (six on average, N = 100; Figure 1B). Non-extracted rad26-GFP cells, prepared in an otherwise identical manner, retained this GFP-fluorescence pattern (data not shown). We also obtained similar results using paraformaldehyde fixation, however in this case non-specific cytoplasmic background fluorescence was higher (data not shown).

Bottom Line: We have discovered three distinct Rad26-GFP cellular structures.Formation of these structures did not require other checkpoint proteins.These data demonstrate that Rad26 can respond to genotoxic insult in the absence of Rad3 and the other checkpoint Rad proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. wolkow@rascal.med.harvard.edu

ABSTRACT

Background: The Rad26/Rad3 complex in fission yeast detects genotoxic insults and initiates the cell cycle arrest and recovery activities of the DNA damage checkpoint. To investigate how the Rad26/Rad3 complex performs these functions, we constructed and characterized Rad26-GFP.

Results: Rad26-GFP localized to approximately six nuclear dots in cycling cells. Following treatment with a DNA damaging agent, Rad26-GFP localization changed. Damaged cells contained one or two bright Rad26-GFP spots, in addition to smaller, more numerous Rad26-GFP speckles. Genetic analyses demonstrated that these Rad26-GFP patterns (dots, spots and speckles) were unaffected by mutations in other DNA damage checkpoint genes, including rad3+. Data obtained with our Rad26.T12-GFP fusion protein correlate spots with cell cycle arrest activities and speckles with DNA repair activities. In addition, physiological experiments demonstrated that rad26Delta and rad3Delta alleles confer sensitivity to a microtubule-depolymerizing drug.

Conclusion: We have discovered three distinct Rad26-GFP cellular structures. Formation of these structures did not require other checkpoint proteins. These data demonstrate that Rad26 can respond to genotoxic insult in the absence of Rad3 and the other checkpoint Rad proteins.

Show MeSH
Related in: MedlinePlus