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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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Rad26.T12-GFP spots, but not speckles, form after Bleomycin treatment. Cultures were grown at 30°C in liquid, complete media to O.D. 0.3 and then treated with 5 mU/ml of Bleomycin for 3 hours. Cells were then prepared for microscopy following the Triton X-100 extraction method or the paraformaldehyde fixation method (see Methods). A. Spots and speckles form in Bleomycin-treated, Triton X-100 extracted rad26-GFP cells. rad26-GFP (TE1197) B. Spots, but not speckles, form in Bleomycin-treated, Triton X-100 extracted rad26.T12-GFP cells. rad26.T12-GFP (TE1206) C. Spots, but not speckles, form in paraformaldehyde-fixed rad26.T12-GFP cells. rad26-GFP (TE1197) and rad26.T12-GFP (TE1206) Bar = 5 μm
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Figure 10: Rad26.T12-GFP spots, but not speckles, form after Bleomycin treatment. Cultures were grown at 30°C in liquid, complete media to O.D. 0.3 and then treated with 5 mU/ml of Bleomycin for 3 hours. Cells were then prepared for microscopy following the Triton X-100 extraction method or the paraformaldehyde fixation method (see Methods). A. Spots and speckles form in Bleomycin-treated, Triton X-100 extracted rad26-GFP cells. rad26-GFP (TE1197) B. Spots, but not speckles, form in Bleomycin-treated, Triton X-100 extracted rad26.T12-GFP cells. rad26.T12-GFP (TE1206) C. Spots, but not speckles, form in paraformaldehyde-fixed rad26.T12-GFP cells. rad26-GFP (TE1197) and rad26.T12-GFP (TE1206) Bar = 5 μm

Mentions: Rad26 functions in checkpoint cell cycle arrest and recovery pathways. A mutant allele, rad26.T12, genetically separates these functions, since rad26.T12 cells can undergo checkpoint-induced cell cycle arrest but fail to recover and lose viability [13,40]. To determine if spots and speckles are associated with these different Rad26 functions, we tagged the C-terminus of Rad26.T12 with GFP using the same primers and method that we used to tag Rad26 (see Methods). After confirming that the rad26.T12-GFP strain behaved similarly to a rad26.T12 strain in response to both Bleomycin and HU (data not shown), we investigated Rad26.T12-GFP localization following Bleomycin treatment. We observed that bright spots formed in both rad26-GFP and rad26.T12-GFP cells that had been extracted with Triton X-100 prior to microscopy (Figure 9, Figure 10A and 10B). We also observed speckles in rad26-GFP cells (Figure 10A) but did not identify these structures in Bleomycin-treated rad26.T12-GFP cells (Figure 10B). Likewise, both spots and speckles formed in paraformaldehyde-fixed rad26-GFP cells, while only spots formed in rad26.T12-GFP cells (Figure 10C). These data link the bright, Rad26-GFP spots with the cell cycle arrest activities of the DNA damage checkpoint, and suggest that Rad26-GFP speckles play a role in recovery.


Fission yeast Rad26 responds to DNA damage independently of Rad3.

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

Rad26.T12-GFP spots, but not speckles, form after Bleomycin treatment. Cultures were grown at 30°C in liquid, complete media to O.D. 0.3 and then treated with 5 mU/ml of Bleomycin for 3 hours. Cells were then prepared for microscopy following the Triton X-100 extraction method or the paraformaldehyde fixation method (see Methods). A. Spots and speckles form in Bleomycin-treated, Triton X-100 extracted rad26-GFP cells. rad26-GFP (TE1197) B. Spots, but not speckles, form in Bleomycin-treated, Triton X-100 extracted rad26.T12-GFP cells. rad26.T12-GFP (TE1206) C. Spots, but not speckles, form in paraformaldehyde-fixed rad26.T12-GFP cells. rad26-GFP (TE1197) and rad26.T12-GFP (TE1206) Bar = 5 μm
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Figure 10: Rad26.T12-GFP spots, but not speckles, form after Bleomycin treatment. Cultures were grown at 30°C in liquid, complete media to O.D. 0.3 and then treated with 5 mU/ml of Bleomycin for 3 hours. Cells were then prepared for microscopy following the Triton X-100 extraction method or the paraformaldehyde fixation method (see Methods). A. Spots and speckles form in Bleomycin-treated, Triton X-100 extracted rad26-GFP cells. rad26-GFP (TE1197) B. Spots, but not speckles, form in Bleomycin-treated, Triton X-100 extracted rad26.T12-GFP cells. rad26.T12-GFP (TE1206) C. Spots, but not speckles, form in paraformaldehyde-fixed rad26.T12-GFP cells. rad26-GFP (TE1197) and rad26.T12-GFP (TE1206) Bar = 5 μm
Mentions: Rad26 functions in checkpoint cell cycle arrest and recovery pathways. A mutant allele, rad26.T12, genetically separates these functions, since rad26.T12 cells can undergo checkpoint-induced cell cycle arrest but fail to recover and lose viability [13,40]. To determine if spots and speckles are associated with these different Rad26 functions, we tagged the C-terminus of Rad26.T12 with GFP using the same primers and method that we used to tag Rad26 (see Methods). After confirming that the rad26.T12-GFP strain behaved similarly to a rad26.T12 strain in response to both Bleomycin and HU (data not shown), we investigated Rad26.T12-GFP localization following Bleomycin treatment. We observed that bright spots formed in both rad26-GFP and rad26.T12-GFP cells that had been extracted with Triton X-100 prior to microscopy (Figure 9, Figure 10A and 10B). We also observed speckles in rad26-GFP cells (Figure 10A) but did not identify these structures in Bleomycin-treated rad26.T12-GFP cells (Figure 10B). Likewise, both spots and speckles formed in paraformaldehyde-fixed rad26-GFP cells, while only spots formed in rad26.T12-GFP cells (Figure 10C). These data link the bright, Rad26-GFP spots with the cell cycle arrest activities of the DNA damage checkpoint, and suggest that Rad26-GFP speckles play a role in recovery.

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