1. The Yeast CDK Inhibitor Sic1 Prevents Genomic Instability by Promoting Replication Origin Licensing in Late G1 
Armelle Lengronne, Etienne Schwob 
Molecular Cell 
Volume 9, Issue 5, Pages (May 2002)
DOI: /S (02)

2. Figure 1 Cells Lacking Sic1 Accumulate in Early Mitosis
(A) GAL-SIC1HA1 (E304, WT) and sic1 GAL-SIC1HA1 (E306, sic1Δ) cells were grown in YEP 1% raff + 0.3% gal, then shifted to YEPD for 2 hr to deplete Sic1 and stained with DAPI to determine nuclear morphology.
(B) Spindle morphology in asynchronous TUB1-GFP (E1122; top) and TUB1-GFP sic1 cells (E1157; bottom).
(C) Distances between GFP dots were determined in asynchronous cultures of SPC42-GFP (E472) and SPC42-GFP sic1 (E903) cells.
(D) Small unbudded TUB1-GFP (E1122; top) and TUB1-GFP sic1 (E1157; bottom) cells were isolated by centrifugal elutriation and incubated (t = 0) in SM medium at 30°C. The percentage of short (<2 μm, metaphase, open circles), mid-anaphase (2–4 μm, closed triangles), and long (>4 μm, telophase, open triangles) spindles was determined from fixed cells. Budding index (closed circles) and DNA content (FACS) are indicated as a measure of cell cycle progression.
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2 Partial Sister Chromatid Separation in sic1 Mutants
(A) Wild-type (E885) and sic1 (E900) cells were synchronized by elutriation and reinoculated in SM + Glu medium at 30°C. Budding index (circles) and the percentage of cells with partially (<4 μm, closed triangles) or fully separated (>4 μm, open triangles) chromosome 3 arms were determined.
(B) Partial sister separation is not due to cohesion defects. sic1 cells (E1148) synchronized in G1 by elutriation were reinoculated at 30°C in YEPD medium with (open symbols) or without 15 μg/ml nocodazole (Noc). Budding index (circles) and % cells with separated sisters (triangles) are shown.
(C) Deletion of PDS1 does not suppress sic1's nuclear division and M exit defects. Elutriatiated wild-type (E1000), sic1 (E715), and sic1 pds1 (E945) cells were incubated in YEPD medium at 25°C. Budding index and % cells with DNA stretched through the bud neck are shown.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3
Minichromosome Loss and Mitotic Exit Defects of sic1 Mutants Are Rescued by Deletion of CLB5 and CLB6
(A) Wild-type (K5144), sic1 (E302), sic1 clb5 clb6 (E349), and clb5 clb6 (E348) cells were transformed with either pDK243 (1 ARS) or pDK368-7 (+7 ARS) plasmids. Mitotic loss rates measured, as indicated in Experimental Procedures, are mean values from three independent experiments.
(B) FACS profiles of asynchronously growing wild-type (E001), sic1 (E211), sic1 clb5 clb6 (E288), and sic1 clb2 (E362) cells.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4 Origin-Firing Defects and Protracted S Phase in sic1 Mutants
(A) 3H-uracil incorporation in DNA was measured in TK+ wild-type (E1000; open circles) and sic1 (E715) cells synchronized in G1 by centrifugal elutriation and released at 25°C in SC-Ura medium containing 20 μCi/ml 3H-uracil. Values are the mean of three experiments.
(B) S phase completion is delayed in sic1 mutants. Elutriated TK+ wild-type (E1000; open circles) and sic1 (E715) cells were incubated at 25°C in SC medium containing 0.4 mg/ml BrdU. S phase completion is determined by FluorImager quantitation of BrdU-labeled chromosomes that reenter the pulsed-field gel. Percent unbudded cells indicate synchrony of both cultures (x, wild-type; ▵, sic1).
(C) 2D gel analysis of ARS305, ARS306, ARS501, and ARS1 in wild-type (E001) and sic1 cells (E211). The ratio of bubble arc (arrowhead) to Y arc (arrow) is a measure of firing efficiency. Right, schematic of replication intermediates.
(D) Increased interorigin distances in sic1 mutants. Examples of raw images showing BrdU substitution along single DNA molecules from elutriated TK+ sic1 cells (E715) arrested in early S with HU (30°C) and displayed by molecular combing. Bar is 20 kb. See Lengronne et al. (2001) for details and wild-type images.
(E) Histogram of center-to-center distances between adjacent BrdU signals on combed DNA molecules. Mean interorigin distance (SD) is indicated in each panel.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5
Sic1 Is Essential when preRC Formation Is Restricted to Late G1
(A) 500 cells of wild-type (E001), sic1 GAL-SIC1 (E306), cdc6Δ HO-CDC6 GAL-CDC6 ash1-61 sic1 (E451), and cdc6Δ HO-CDC6 GAL-CDC6 ash1-61 (E453) were spotted on YEPGal and YEPD (Glu) plates and incubated 3 days at 30°C.
(B) HO-CDC6 sic1 cells arrest with 1C DNA. HO-CDC6 (E453) and HO-CDC6 sic1 (E451) cells grown in YEP 1% Raf + 0.3% Gal were transferred to YEPD and analyzed for DNA content (FACS) every hour thereafter. Sample shown is at 2 hr after shift to glucose.
(C) SIC1 interacts with replication initiation mutants. Wild-type (E001), cdc6-1 (E589), cdc14-3 (E590), cdc15-2 (E271), mcm2-1 (E591), mcm3-1 (E488), and orc5-1 (E592) cells, or derivative thereof (bottom row), also containing sic1 and GAL-SIC1HA (E306, E356, E368, E366, E524, E593, and E529 strains, respectively) were grown in YEP 1% Raf + 0.3% Gal at 25°C and spotted onto YEPD (left) or YEP 1% sucrose + 1% Gal (right) plates to deplete or moderately overexpress Sic1, respectively. Plates were incubated 2 days at 25°C (top) or 30°C (bottom) and photographed.
(D) Checkpoint independent mitotic delay. Wild-type (E1000), sic1 (E1148), sic1 rad9 (E1172), sic1 mec1-1 (E1062), sic1 mad1 (E708), and sic1 bub2 (E1263) cells were synchronized by elutriation and incubated at 30°C. FACS profiles of the corresponding cultures are shown.
(E) Slow growth phenotype. Serial 5-fold dilutions of wild-type (E001), sic1 (E210), rad9 (E231), sic1 rad9 (E1172), rad24 (E1420), sic1 rad24 (E1419), ddc1 (E1458), sic1 ddc1 (E1486), mec1-1 (E73), sic1 mec1-1 (E236), pol2-12 (E1460), sic1 pol2-12 (E1489), rfc5-1 (E1462), and sic1 rfc5-1 (E1492) cells were spotted on YEPD and incubated 48 hr at 30°C. mrc1Δ showed no effect (data not shown).
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6 DNA Damage and Gross Chromosomal Rearrangements
(A) Log phase cultures of DDC1-GFP (E1528) and sic1 DDC1-GFP (E1565) cells were analyzed by fluorescence microscopy. Left, arrows indicate Ddc1-GFP foci in sic1 cells. Right, number of cells with Ddc1-GFP foci.
(B) Accumulation of Ddc1 foci in sic1 cells depends on anaphase entry. Early G1 sic1 DDC1-GFP (E1565) cells were incubated in YEPD medium at 30°C. At t = 90 min, α factor was added to the culture which was then split in two, one half receiving 15 μg/ml nocodazole. Left, fraction of cells containing Ddc1-GFP foci at t = 0 and t = 210 min (±Noc). Right, FACS profiles of the corresponding cultures are shown.
(C) Genomic instability. Chromosomes from Canr-FoAr clones isolated from strain E1601 (sic1 CAN1 hxt13::URA3) were resolved by PFGE and stained with Sybr-Gold (lanes 1–20). The parental Cans-FoAs strain (E1557) was used as control (lanes marked C).
(D) Southern hybridization of the gel shown in (C) with a probe (MCM3) of Chr.5. Arrows indicate Chr.5 arm loss events followed by telomere addition. Chromosome number and size are indicated left and right, respectively.
Molecular Cell 2002 9, DOI: ( /S (02) )