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. 2016 Oct 25;7:13157. doi: 10.1038/ncomms13157

Figure 2. Dna2 helicase-defective cells suffer dual growth inhibition by checkpoint activation and loss of YEN1.

Figure 2

(a) Cell viability of the indicated strains assessed by colony outgrowth. The mean plating efficiency (PE)±s.e.m. (n=3) is presented relative to wild-type. (b) Microscopic analysis of the indicated strains growing exponentially in rich medium. Left, representative images (DIC) showing morphological changes associated with Dna2 helicase dysfunction and loss of Yen1. Average doubling times are given (n=3). Scale bar, 5 μm. Centre, mean distribution of G1, S, and G2/M cells±s.e.m. (n=4). Right, DNA segregation in G2/M cells as determined by DAPI-staining. An average of 200 cells were scored per strain. (c) Flow cytometric analysis of the indicated strains synchronized in G1 using α-factor and released into YPAD. The position of cells with 1 and 2 N DNA content is indicated. Topmost tracks are asynchronous cultures overlaid with the outline of the wild-type profile, showing that Dna2 helicase-defective strains accumulate cells with a 2 N content over time. (d) Western blot analysis showing chronic low-level DNA damage checkpoint activation in Dna2 helicase-defective cells as indicated by Rad53 phosphorylation (Rad53-P). (e) Effect of DNA damage checkpoint disruption by deletion of RAD9 on the distribution of G1, S, and G2/M cells in exponentially growing cultures of the indicated strains. Data presented as mean distribution±s.e.m. (n=3). (f) Cell viability of the indicated strains assessed by colony outgrowth. The mean plating efficiency±s.e.m. (n=3) is presented relative to a rad9Δ control.