Figure 5. Forced accumulation of replisome components in swi1Δ cells causes catastrophic DNA replication and mitotic abnormalities.

(A) swi1Δ pof3Δ cells have increased levels of mitotic catastrophes. Exponentially growing cells were treated with or without the indicated drugs (12 mM HU or 20 µM CPT for 6 h), fixed in ethanol, and stained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of observed nuclear phenotypes are shown. Representative mitotic failures are shown by arrows. Arrows were omitted from the images of swi1Δ pof3Δ cells because a large numbers of cells showed mitotic catastrophes. The scale bar represents 10 µM. (B) Quantification of cells with defective mitosis including chromosome missegregation, aneuploidy, cut and other aberrant phenotypes. More than 200 cells were counted for each strain. Error bars correspond to standard deviations obtained from three experiments. (C) DNA damage sensitivity of swi1Δ mutants is increased by pof3 deletion. Five-fold serial dilutions of cells were incubated on YES agar medium supplemented with the indicated drugs (2 mM HU or 1 µM CPT) for 2 to 3 days at 32°C. (D) pof3 deletion exacerbates replication recovery defects of swi1Δ mutants. Exponentially growing cells (Log) were incubated in the presence of 5 µM CPT for 3 h at 30°C (CPT), then washed and returned into fresh medium for 2 h or 4 h (2 h, 4 h). Chromosome samples were examined by PFGE. Representative results of repeat experiments are shown. swi1Δ cells have shorter chromosome III due to hyper recombination at rDNA repeats [42], [70], [101].