Figure 1.
Survivors of the untimely initiation of DNA replication experiments have aberrant chromosomes.
A, Chromosomal DNA from YST563 (JET1-1 GALp-sld2-11D) survivors from Figure S1 was analyzed using pulsed-field gel electrophoresis. Surviving colonies (survivors 1–17) were recovered on YPAD plates after galactose incubation. Abnormal chromosome bands are indicated with arrowheads, chromosomes with increased band intensity are indicated with filled arrowheads, and chromosomes with different lengths are indicated with open arrowheads. Chr XII bands are not marked because they are unstable even in wild-type cells. B, Quantified profiles of band intensities of A are shown. Abnormal chromosome bands are indicated with arrowheads. *: non-chromosomal signal originated from contaminated dusts.
Figure 2.
Endogenous levels of Sld2-11D do not promote untimely DNA replication efficiently.
A, W303-1a Δbar1 (wt), YST556 (JET1-1), YST831 (sld2-11D), YST816 (JET1-1 sld2-11D), YST562 (JET1-1 GALp-sld2-11D) and YST560 (JET1-1 GALp-SLD2) were grown on YPAD (Glc) or YPAGal (Gal). B, YST827 (JET1-1 SLD2 (wt)) and YST829 (JET1-1 sld2-11D) cells were grown in YPARaffinose medium (Asyn), arrested in G1 phase with alpha factor, galactose was added and samples were taken at the indicated times. The DNA contents of the samples were analyzed by flow cytometry. C, Small aliquots of the same samples from B were spread onto YPAD plates, and the viability was calculated from the number of colonies that appeared after incubation. D, Whole cell extracts were prepared from the same samples from B and were analyzed by western blotting. Sld2 and Orc6 proteins were detected with anti-Sld2 and anti-Orc6 antibodies, respectively. The loading control shows the corresponding region of the Ponceau-S-stained membrane.
Figure 3.
G1/S-level Sld2-11D can promote DNA replication.
A, YST1332 (GALp-SIC1ΔNT CDC45 (wt) SLD2 (wt)) YST827 (GALp-SIC1ΔNT JET1-1 SLD2 (wt)) and YST829 (GALp-SIC1ΔNT JET1-1 sld2-11D) cells were grown in YPARaffinose medium (Asyn) and arrested in G1 phase with alpha factor (α). Galactose was added, and cells were incubated for 1 hour to express Sic1ΔNT. Cells were then released into fresh YPAGal and collected at the indicated times after release (0–300 min). The samples were analyzed by flow cytometry. B, The proportion of budded cells (YST1332: open boxes; YST827: open circles; YST829: filled circles) at the indicated times are shown. C, Whole cell extract was prepared from the same samples as A and analyzed by western blotting. Sld2 proteins and Orc6 were detected with anti-Sld2 and anti-Orc6 antibodies, respectively. The loading control shows the corresponding region of the Ponceau-S-stained membrane.
Figure 4.
The Sld2-11D protein level is important to promote DNA replication.
A, YST1698 (JET1-1 GALp-sld2-11D ORC6-FLAG) cells were grown in YPARaffinose medium (Asyn) and arrested in G1 phase with alpha factor, and then the culture was split into six portions. Different amounts of galactose were added to each portion, and samples were taken at the indicated times (0–6 hours after galactose addition). The DNA contents of the samples were analyzed by flow cytometry. B, DNA content at 0, 4 and 6 hours of different galactose amounts are compared by overlay. C, Whole cell extracts were prepared from the same samples from A and analyzed by western blotting. Sld2 proteins and Orc6-FLAG proteins were detected with anti-Sld2 and anti-FLAG antibodies, respectively. *: non-specific background band.
Figure 5.
High-level expression of Sld2 in G1 induces GCR.
A, Relative GCR values in Table 1 are graphically shown. The GCR rate of YST1007 (GALp vector) grown in galactose-containing medium is set as one. B, YST1007 (vector), YST1008 (SLD2) and YST1024 (sld2-11D) cells were grown in YPAD (OFF) or YPAGal (ON) medium (Asynchronous: As), and aliquots of the YPAGal culture were arrested in G1, S or G2/M phase with alpha factor, HU or nocodazole, respectively. Cells were collected, and Sld2 proteins were detected with anti-Sld2 antibody. The loading control shows the corresponding region of the Ponceau-S-stained membrane. C, YST1338 (Db vector), YST1062 (Db-SLD2) and YST1064 (Db-sld2-11D) cells were grown and analyzed as in B. The Db fragment (Myc-tagged) was expressed from the Db vector and detected with anti-Myc antibody. *: non-specific background bands. D, YST1159 (sic1N vector), YST1161 (sic1N-SLD2) and YST1162 (sic1N-sld2-11D) cells were grown and analyzed as in B. The Sic1N fragment (Myc-tagged) was expressed from the sic1N vector and detected with anti-Myc antibody.
Table 1.
The GCR rate for GALp-SLD2 cells.
Figure 6.
Extra Sld2-11D expression similar at endogenous level can induce GCR.
A, Relative GCR values in Table 2 are graphically shown. The GCR rate of RDKY3615 (Wt (SLD2)) is set as one. B, RDKY3615 (Wt (SLD2)), YST1336 (sld2-11D), YST1090 (+vector), YST1737 (+SLD2), YST1739 (+sld2-11D), YST1747 (+sic1N-SLD2) and YST1749 (sic1N-sld2-11D) cells were grown (asynchronous), and aliquots of the culture were arrested in G1 or S phase with alpha factor or HU, respectively. Whole cell extracts were prepared and subjected to western blotting. Sld2 proteins were detected with anti-Sld2 antibody. The loading control shows the corresponding region of the Ponceau-S-stained membrane. *: non-specific background band. Lanes 14, 15, 21 and 22 are enlarged to show the Sic1N-Sld2 bands (bottom).
Table 2.
The GCR rate of cells with extra copies of SLD2.
Figure 7.
Untimely DNA replication conferred the accumulation of phosphorylated Rad53 and Ddc1 foci after S phase.
A, YST1700 cells (CDC45JET1-1 GALp-sld2-11D DDC1-GFP) were grown in YPARaffinose medium (Asyn) and arrested in G1 phase with alpha factor (α). The culture was then split into two portions, and galactose was added to one portion (Gal (ON)) and further incubated for 2 hours. Then cells were released into fresh YPAD, and samples were taken at the indicated times (0–120 min). The DNA contents of the samples were analyzed by flow cytometry. B, Whole cell extracts were prepared from the same samples from A and subjected to western blotting. Rad53 protein was detected with anti-Rad53 antibody. *: non-specific background band. C, D, YST1700 cells were grown in SC-Sucrose medium, arrested in G1 and split into two portions, and galactose was added to one portion (Gal (ON)) and released into fresh SC-glucose. Samples were taken at the indicated times (0–120 min), and Ddc1-GFP was observed under the microscope (C). The proportion of cells with Ddc1-foci or bud was counted (D).
Table 3.
Strains.