Reciprocal shift analysis of ChrXII mobility. sgs1-34 slx4Δ cells were arrested with α-factor at 25°C (25/αF) and released at 37°C in the presence of nocodazole (37/noc) for three hours. The cells were then washed and released at 25°C in the absence (25/ypd) or presence (25/αF) of α-factor for three hours. After the indicated treatments cells were harvested and DNA was analyzed by CHEF gel electrophoresis. A Ethidium bromide stain. B Southern blot with rDNA probe.

Extra-chromosomal rDNA circle formation in sgs1Δ and slxΔ strains. Total DNA was isolated from sorted seven-generation old cells and unsorted young cells. This DNA was subjected to one-dimensional gel electrophoresis and Southern blotted using an rDNA fragment as probe. The average bud scar counts of sorted cells was determined to be between 7 and 8. A control strain (C; top1 top2–4) excises rDNA circles at 25°C and is used as a marker for ERC migration. Y, young cells; O, old cells.

Altered electrophoretic mobility of ChrXII in sgs1-ts slx4Δ strains. The indicated strains were grown to exponential phase at 25°C, arrested with α-factor and released at 37°C for the indicated times. Cells were collected and prepared for CHEF gel electrophoresis in agarose plugs. The gel was stained with ethidium bromide (top) and Southern blotted using an rDNA fragment as probe (bottom). The bottom half of the blot had no signal and is not presented. The large arrowhead indicates the position of ChrXII and the small arrowhead indicates the position of the wells.

Multiple rDNA repeat units are required for the altered electrophoretic mobility of ChrXII. The sgs1-34 slx4 strain was grown to exponential phase at 25°C, arrested with α-factor and released at 37° for 6 hours. Cells were collected and prepared for CHEF gel electrophoresis in agarose plugs. The plugs were digested with NotI or SacI as indicated. A Following electrophoresis the gel was blotted using the complete TOP3 coding region (left) or an rDNA fragment (right) as probe. B The electrophoresis of chromosomal DNA was performed as above except that the electrophoresis was stopped after 7 hours. The gel was then Southern blotted using an rDNA fragment as probe.

Cell cycle arrest of sgs1-ts slx4Δ cells. The indicated strains were grown to exponential phase at 25°C and synchronized by arresting growth with α-factor. After release into YPD broth at 37°C, aliquots were removed at the indicated times and fixed in ethanol. A One portion of each aliquot was stained with propidium iodide and analyzed by flow cytometery. B Another portion was microscopically examined for budding morphology: gray, single cells; crosshatch, small budded cells; white, large budded cells; black, aberrant or multiply-budded cells. Data is presented as a percentage of at least 100 cells.

Conditional SGS1 activity. A Strains of the indicated genotype were grown to exponential phase in YPD broth at 25°C, transferred to 37°C for the indicated times, and the percentage of viable cells determined. B Recombination rate was determined by stably transforming the indicated SGS1 allele, or vector alone, into strain NJY540 (sgs1::loxP) which contains the URA3 gene integrated at RDN1. The number of uracil auxotrophs was determined following 3 days growth in the absence of selection at the indicated temperature. The percentage of cells undergoing a marker loss event was determined and is presented as the mean ± SD from seven independent colonies. C SGS1-dependent hypersensitivity to DNA damaging agents was determined by resuspending strains of the indicated genotype and spotting them in 10-fold serial dilutions on YPD plates containing MMS or no drug. The plates were photographed following three days at the indicated temperature.

Replication of the rDNA is required for the altered mobility of ChrXII in sgs1-ts slx4Δ cells. A Cells were synchronized and analyzed as in Fig 3. Left panel: the sgs1Δ and sgs1-34 single mutants were released from αF block and incubated for 6 hours in YPD broth at the indicated temperature. Right panel: the indicated double mutants were released into medium containing α-factor or nocodazole and incubated for 3 hrs at the indicated temperature. Microscopic examination confirmed that 98% of the cells held in α-factor were unbudded and 97% of the cells blocked in nocodazole were large-budded. Cells were harvested and analyzed by CHEF gel electrophoresis. The large arrowhead indicates the position of ChrXII and the small arrowhead indicates the position of the wells. B Density shift analysis of rDNA replication. sgs1-34 slx4Δ cells were grown in heavy minimal medium and arrested with α-factor at 25°C (αF/25). Cells were then washed, and aliquots released at 25°C into light medium (No Drug/25) or at 37°C into light medium containing α-factor (αF/37) or nocodazole (Noc/37). After the indicated treatments the cells were harvested and the DNA was isolated, fractionated by equilibrium gradient density sedimentation, and analyzed by Southern blotting. The positions of unreplicated heavy-heavy (HH) and fully replicated heavy-light DNA (HL) are indicated. The initial HH peak is shown for comparison as a shaded peak in the released samples.