Prevention of nuclear localization of the Mcm2-7 complex by Cln2 is important for inhibition of DNA rereplication. (A) Cln2 or Cln2-1 expression blocks nuclear accumulation of Mcm2-7. YST66 (+CLN2), YST67 (+CLN2-1), and YST69 (+vector) were grown and treated as in Figure 3 except that samples were taken after either 2 or 3 h in YP-galactose. Samples were prepared for microscopy to examine which cells showed nuclear accumulation of Mcm4–GFP. (B) Cln2 or Cln2-1 expression cannot prevent rereplication when Cdk inhibition of Cdc6 and Mcm2-7 is bypassed. YST282 (+CLN2 MCM7-2NLS), YST283 (+CLN2 MCM7-2nls3A), YST284 (+CLN2-1 MCM7-2NLS), YST285 (+CLN2-1 MCM7-2nls3A), YST286 (+vector MCM7-2NLS), and YST287 (+vector MCM7-2nls3A) were grown as in Figure 2. Cells were collected and analyzed by flow cytometry.

Cln2 expression can induce genomic instability. (A) Genome instability caused by Cln2 expression. An aliquot of the culture from the last time point of the rereplication assay in Figure 2C was spread onto fresh YP-glucose plates, and survivors were recovered. Each colony was grown in YP-glucose medium, fixed, and analyzed by flow cytometry to examine the ploidy. (B) Cln2 or Cln2-1 induces GCR. The GCR rates of YST196 (vector), YST192 (CLN2), and YST193 (CLN2-1) were measured as described in Materials and Methods. The ratios of the GCR rate between growth in YP-galactose and YP-glucose for each strain are shown. (C) Cln2-1-induced GCR is suppressed by multiple origins. The GCR rates of YST248 (vector) and YST252 (7× ARS) were measured as described in Materials and Methods. The ratios of the GCR rate between growth in YP-galactose and YP-glucose for each strain are shown.

Cln2 can inhibit pre-RC formation. DNase I genomic footprinting. YST67 (+CLN2-1) and YST69 (+vector) were grown in YP-raffinose to early log phase at 24°C. One-third of the culture was arrested at G₁ in α-factor for 3.5 h (α). The remaining culture was arrested in G₂/M with nocodazole for 3 h (noc), after which the medium of half of the culture was changed to YP-galactose and incubated at 24°C for a further 4 h (Gal). Genomic DNA was prepared, and primer–extension reactions were performed with an ARS305-specific primer. G₁-specific DNase I hypersensitive sites (diamond), protection (black box), and G₂-specific hypersensitive sites (*) are shown.

Cln2 overexpression induces plasmid loss that is suppressed by multiple origins. (A) Histone H1 kinase activity after Cln2 or Cln2-1 induction. YST66 (+CLN2), YST67 (+CLN2-1), and YST69 (+vector) were grown in YP-raffinose. Each culture was split into two, and the medium was changed to YP-galactose (Induction; +) or YP-glucose (Induction; −). Cells were collected after a 60-min incubation, and Myc-tagged Cln2 and Cln2-1 were immunoprecipitated with 9E11 anti-Myc antibody (9E11) or without the antibody (mock). The H1 kinase activity of the immunoprecipitates was measured and compared between the samples before and after the medium change. (B) Elevated plasmid loss rates after Cn2 or Cln2-1 expression. YST177 (CLN2, pDK368-1), YST178 (CLN2, pDK368-7), YST181 (CLN2-1, pDK368-1), and YST182 (CLN2-1, pDK368-7) were grown as described in Materials and Methods, and the rate of plasmid loss from each strain was measured. Plasmid loss rates after growing the cells in Cln-inductive and Cln-repressive conditions are shown by gray and black boxes, respectively.

Cln2 can inhibit DNA rereplication induced by Sic1C70td. (A) Temperature-dependent proteolysis of Sic1C70td. YST78 [wild type (wt), +SIC1C70td], YST48 (wt, +SIC1C70td, UBR1), YLD11 (wt, +SIC1), YST71 (cdc4-1, +SIC1C70td), YST76 (cdc4-1, +SIC1C70td, UBR1), and YST27 (cdc4-1, +SIC1) were grown in YP-raffinose to early log phase (asyn) and arrested in G₂/M with nocodazole (noc) at 24°C for 3 h. Then the medium was changed to YP-galactose plus nocodazole, and the cells were incubated for 2 h. The culture was then split into two, the medium was changed to YP-glucose plus nocodazole, and the cultures were incubated at 24°C or 37°C. Cells were collected every 30 min (0–120 min) and processed for immunoblotting. Sic1C70td–Myc, Sic1–Myc, and Myc–Ubr1 (data not shown) were detected with 9E10 anti-Myc antibody. (LC) Loading control. (B) Transient Sic1C70td expression induces rereplication in nocodazole-arrested cells. YST65 (+SIC1C70td), YST64 (+SIC1ΔNT), and YST57 (+vector) were grown as in A. Cells were collected at each time point and processed for flow cytometry. (C,D) Cln2 and Cln2-1 expression inhibits Sic1C70td-induced rereplication. YST66 (+CLN2), YST67 (+CLN2-1), and YST69 (+vector) were grown, and cells were collected as in A. Each sample was analyzed by flow cytometry (C) or immunoblotting (D). The second largest subunits of DNA polymerase I (p86/91), Orc6, Cln2–Myc, Cln2-1–Myc, and Sic1C70td–Myc were detected by probing with the appropriate antibodies. The ladder bands commonly observed in lanes 3, 8, and 13 of the Cln2/Cln2-1 panel are ubiquitinated Sic1C70td. LC, loading control from the PonceauS-stained membrane.