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. 2011 Aug 25;7(8):e1002263. doi: 10.1371/journal.pgen.1002263

Figure 1. SSA-mediated DSB repair in yku70Δ G1 cells.

Figure 1

(A) Map of the YMV86 chromosome III region where the HO-cut site is flanked by homologous leu2 sequences that are 0.7 kb apart. HO-induced DSB formation results in generation of 3.2 kb and 1.8 kb DNA fragments (HO-cut) that can be detected by Southern blot analysis of BglII-digested genomic DNA with a LEU2 probe. DSB repair by SSA generates a product of 2.9 kb (SSA product). B, BglII. (B–E) Exponentially growing YEP+raf (exp) cell cultures of wild type YMV86 and its yku70Δ derivative strain were arrested in G1 with α-factor (time zero) and transferred to YEP+raf+gal in the presence of α-factor. (B) FACS analysis of DNA content. (C) Southern blot analysis of BglII-digested genomic DNA. (D, E) Densitometric analysis of the HO-cut (D) and the SSA (E) band signals. Plotted values are the mean value ±SD from four independent experiments as in (C), enclosing that described in (F). The intensity of each band was normalized with respect to a loading control. (F) YMV86 derivative strains with the indicated genotypes and expressing fully functional Cdc28-HA were treated as in (B–E). Cell samples were collected at the indicated times to assay Cdk1 kinase activity in anti-HA immunoprecipitates by using histone H1 as substrate (top row) and to determine Cdk1 levels by western blot analysis with anti-HA antibody (bottom row).