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First published online 11 December 2002
doi: 10.1242/jcs.00251

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Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae

Douglas A. Thrower*, Jennifer Stemple, Elaine Yeh and Kerry Bloom{ddagger}

Department of Biology, CB3280 University of North Carolina, Chapel Hill, NC 27599-3280, USA
* Present address: Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Barbara, Santa Barbara, CA 93106, USA



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  		Fig. 1. Homologous repair of a dicentric chromosome in budding yeast requires RAD52 and RAD1 but not RAD9. (A) The diagram shows locations of BamHI restriction sites (labeled `B') flanking the endogenous CEN3, GALCEN3 (upper diagram) and CEN3-GALCEN3 recombination products (lower diagram). (B) Southern analysis of BamHI-digested genomic DNA hybridized to a labeled CEN3 fragment reveals the loss of a 0.865 kb GALCEN3 fragment (arrow) and the formation of a 1.1 kb fragment (arrowhead) following a shift from galactose to glucose to activate the dicentric chromosome. No recombination product is observed in a rad52 deletion strain (C) nor in a rad1 deletion strain (D). The kinetics of CEN3-GALCEN3 recombination are unaffected by deletion of RAD9 (E). The numbers below lanes refer to the time of sample collection, in hours, following transfer of cells to glucose. The positions of molecular weight markers (kb) are indicated.

 


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  		Fig. 2. RAD52-independent homologous repair of a dicentric chromosome in cells containing the cdc5-ad allele requires RAD1 and RAD9. (A) Southern analysis of CEN3-GALCEN recombination in a cdc5-ad strain following activation of a dicentric chromosome. (B) CEN3-GALCEN recombination also occurs in a cdc5-ad rad52 strain following activation of a dicentric chromosome but this recombination product is not detected in a cdc5-ad rad1 rad52 double deletion strain (C) or in a cdc5-ad rad9 rad52 strain (D). The arrow indicates the position of the original band and the arrowhead indicates the recombinant band. The numbers below the lanes refer to time of sample collection, in hours, following transfer of strains from galactose to glucose. The positions of molecular weight markers (kb) are indicated.

 


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  		Fig. 3. Changes in nuclear morphology and repeated nuclear oscillations result from activation of a dicentric chromosome in cdc5-ad rad52 cells. (A-Q) Sequential fluorescence images of histone2B-GFP-labeled nuclei in a dicentric cdc5-ad rad52 cell. (R) DIC image of cell in A-Q. The time after activation of dicentric chromosome is indicated (hours, minutes) in the lower right corner of each panel. Bar, 5 µm.

 


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  		Fig. 4. Requirement for microtubules but not dynein for nuclear dynamics in dicentric cdc5-ad rad52 cells. (A-D) Sequential fluorescence images of a dicentic cdc5-ad rad52 cell expressing histone2B-GFP following treatment with benomyl. (E-H) Sequential fluorescence images of two dicentric cdc5-ad dhc1 rad52 cells expressing histone2B-GFP. Note the change in nuclear morphology of the right-hand cell from round with a tail to `dumbbell' (Fig. 4E,F). Also note the growth of filaments from both nuclei, indicated by arrows in Fig. 4G,H. Elapsed time is indicated in seconds. Bar, 5 µm.

 


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  		Fig. 5. Suppression of nuclear filament dynamics in dicentric cdc5-ad rad52 cells by ATP depletion. Life history plot of a nuclear filament before, during and after removal of sodium azide and deoxyglucose. Media containing azide and deoxyglucose was added after 23 minutes and replaced by with drug-free media after 58 minutes.

 


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  		Fig. 6. Viability of cells containing either an inverted or a direct repeat dicentric chromosome is inhibited by low concentrations of benomyl. Strains containing either inverted or direct repeat dicentric constructs were grown in galactose media, washed and plated on solid media containing either galactose or glucose and the indicated concentrations of benomyl. The proportion of viable cells was determined by dividing the number of colonies that grew on glucose media by those on galactose. Shown are mean values for the results of three independent experiments, normalized to a control viability of 100%.

 

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© The Company of Biologists Ltd 2003