Cell, Volume 128
Supplemental Data
The Origin Recognition Complex
Functions in Sister-Chromatid Cohesion
in Saccharomyces cerevisiae
Kenji Shimada and Susan M. Gasser
Supplemental Experimental Procedures
Yeast Strains and Cell Culture
Yeast strains used in this study are listed in Table S1. All strains are derivatives of W303-1A. Wild-type and mutant
20xARS1 unit (Kawasaki et al., 2006) were cloned into pRS403 and integrated to the 5’ UAS of URA3 gene to
generate strains harboring ARS1 array. Media was supplemented with 25 mg/L adenine, but otherwise standard media
were used Rose et al., 1990. Whenever nocodazole was added the culture was adjusted to 1% DMSO.
Antibodies
Monoclonal antibodies (mAb) against Orc1 (SB35), Orc2 (SB46), Orc3 (SB3), Orc4 (SB6), Orc5 (SB5), and Orc6
(SB49) were generous gifts of Dr B. Stillman (Liang and Stillman, 1997). Polyclonal antibodies were raised against
holo-topoisomerase II (a2), the N-terminal 173 residues of Orc2, or 275-562 residues of Sir2, and the latter two were
affinity purified before use. Anti-yeast Mcm2 was obtained from Santa Cruz Antibodies (yN-19). Other antibodies
used ware mAb 9E10 (anti-Myc), mAB 12CA5 (anti-HA for Western blot), mAb F-7 (anti-HA for ChIP assay, Santa
Cruz,), and mAb M2 (anti-FLAG, Sigma).
Western Blot Analysis
Western blot detection was carried out by HRP-conjugated secondary antibodies and signal was detected on the X-ray
film. Signal quantification was carried out by direct chemiluminescence acquisition with Quantitiy One software
(BioRad). Antibody dilutions were; affinity-purified anti-Orc2: 1/20, anti-Orc1 (SB35):1/2000, anti-Orc3 (SB35):
1/4000, anti-Orc6 (SB49): 1/4000, anti-MCM2: 1/3000, anti-Top2: 1/7500, anti-HA (12CA5): 1/3000, and anti-Myc
(9E10) 1/5000.
ChIP (Chromatin Immunoprecipitation) Analysis
ChIP was performed with appropriate antibodies coupled with protein G or anti-mouse IgG-Dynal beads (Dynal) on
cells fixed with 1% formaldehyde for 15 min at 30 °C. ORC ChIP was performed with protein G-Dynal beads coupled
with mixture of mABs against Orc1 (SB35), Orc2 (SB46), Orc3 (SB3), and Orc4 (SB6). IP, reverse crosslink, and
DNA recovery procedures were essentially carried out as described in Cobb et al., 2003. Primer sets around ARS501
on Chr 5 (Fig. 4E) were described in Tanaka et al., 1999. Quantitative ChIP analysis was performed using real time
PCR with SYBR green method (Platinum SYBR Green qPCR SuperMix UDG; Invitrogen). To obtain linier
amplification, concentration of each primer set was determined in a serial dilution of genomic DNA. Fold enrichment
of IP-DNA was calculated as enrichment over the FAB1 control locus (14 kb away from ARS607). All experiments
were performed several times, and standard deviation of the mean was based on all available data. Primer sequences
used were;
CEN4
SG-2849: 5’-CTCATGCAATTGCTCAAAGCGTA-3’
SG-2850: 5’-TTACCGCTGTATGCAATTTCTTGTG-3’
YMR31
SG-2851: 5’-ATTAGCAAAGAGTGCATATGAGCCG-3’
SG-2852: 5’-GCAAGGATGAACAACGACCTCAG-3’
SOK1
SG-2860: 5’-GGGAGGGAGATGGATTGTTGATA-3’
SG-2861: 5’-AGACTGGTCAATTCTTGAAAAACGA-3’
FAB1
SG-1895: 5’-CAGGATATGCGGCCAAATTT-3’
SG-1896: 5’-GCATGACAGCCGAATCGAT-3’
ARS1 array
SG-2821: 5’-GCTAAATCATTTGGCTTTGAATTCA-3’
SG-2822: 5’-GACGAAATTTGCTATTTTGGATCC-3’
FACS Analysis
FACS analysis was carried out essentially as described in Frei and Gasser, 2000.
Sister Chromatid Cohesion Assay
Cells harboring LacO or TetO repeats were fixed with 2% paraformaldehyde at room temperature for 10 min, then
washed with PBS for 3 times. GFP signals were recorded in 3D (0.2µm interval, 21 sections) by Till2 wide-field
microscope with Metamorph software. Cells exhibiting 2 GFP spots were counted over 100 cells.
Live Fluorescence Microscopy Analysis
Cultures were grown to 5-8 x 106cells/ml. Cells were trapped on a concanavanin A coated coverslip in a Ludin
chamber (Life Imaging Services) and time-lapse imaging was performed at 30°C using a Zeiss LSM510 confocal
microscope as described in Heun et al., 2001. Settings for the Zeiss LSM510 were as follows: Argon laser 4.7 Amp,
Output 25%, captured on Channel 1 using Lp 505 for GFP alone; detector gain: 930 to 999; amplifier gain: 1-1.5;
amplifier offset: 0.2V–0.1 V; laser transmission AOTF = 2 - 5%; scan speed was 10 (1.28 µs/pixel); 8 bits one scan
direction; 2 average/Mean/Line (or 4 average for SCC1-GFP movie), using a zoom 1.8 (pixel size: 100 x 100 nm).
Under these conditions, cells continued to divide without obvious cell cycle delay suggesting that phototoxic damage
was minimized (Heun et al., 2001).
For the GAL:orc2-1 reinduction experiment, SD-his (glucose medium) was exchanged three times with pre-warmed
(30°C) S-2% galactose-his by gently pumping with the syringe. Time-lapse imaging was continuously performed
before and after the change of media on the same field of cells.
Supplemental References
Cobb, J. A., Bjergbaek, L., Shimada, K., Frei, C., and Gasser, S. M. (2003). DNA polymerase stabilization at stalled
replication forks requires Mec1 and the RecQ helicase Sgs1. Embo J 22, 4325-4336.
Frei, C., and Gasser, S. M. (2000). The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication
checkpoint and colocalizes with Rad53p in S-phase-specific foci. Genes & Development 14, 81-96.
Heun, P., Laroche, T., Shimada, K., Furrer, P., and Gasser, S. M. (2001). Chromosome dynamics in the yeast
interphase nucleus. Science 294, 2181-2186.
Kawasaki, Y., Kim, H. D., Kojima, A., Seki, T., and Sugino, A. (2006). Reconstitution of Saccharomyces cerevisiae
prereplicative complex assembly in vitro. Genes Cells 11, 745-756.
Lengronne, A., Katou, Y., Mori, S., Yokobayashi, S., Kelly, G. P., Itoh, T., Watanabe, Y., Shirahige, K., and Uhlmann,
F. (2004). Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature.
Liang, C., and Stillman, B. (1997). Persistent initiation of DNA replication and chromatin-bound MCM proteins
during the cell cycle in cdc6 mutants. Genes & Development 11, 3375-3386.
Pellicioli, A., Lucca, C., Liberi, G., Marini, F., Lopes, M., Plevani, P., Romano, A., Di Fiore, P. P., and Foiani, M.
(1999). Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the
lagging strand DNA polymerase. EMBO Journal 18, 6561-6572.
Tanaka, T., Cosma, M. P., Wirth, K., and Nasmyth, K. (1999). Identification of cohesin association sites at
centromeres and along chromosome arms. Cell 98, 847-858.
Figure S1. Rad53 Is Transiently Activated in orc2-Depleted Cells in Late S or G2 Phase
Wild-type (GA-1906) and GAL:orc2-1 (GA-1908) were blocked by α factor in YPG then further cultured in YPD + α
factor for 1.5h at 30°C to deplete orc2-1p. Cells were released in fresh YPD at 30°C and samples were taken for Rad53
autophosphorylation assay (A) (Pellicioli et al., 1999) and FACS analysis (B). Autoradiography signal of Rad53
activity was first standardized by Western blot signal (anti-Rad53-13Myc). The graph represents Rad53 activation
relative to wild-type + 0.2M HU signal for100%.
Figure S2. Scc1-18myc ChIP Analysis Probed for SOK1 Locus
A. Scc1-18myc ChIP was performed in wild-type and GAL:orc2-1 cells as described in Fig. 4B. Enrichment of SOK1
signal was normalized by FAB1 control and plotted. Value obtained from wild-type and GAL:orc2-1 cells are 1.09 ±
0.15 and 1.02 ± 0.08, respectively. B. Arrows show positions of CEN4, SOK1, and YMR31 probe (Scc1-localization
map adopted from Lengronne et al., 2004). TRP1 and ARS609 loci are highlighted in red. Standard deviation of the
mean was based on all available data.
Figure S3. Orc2-1 Reinduction in G2-M Rescues Cell Viability
Wild-type (GA-2146), eco1-1 (GA-2825), GAL:orc2-1 (GA-2147) were synchronously released into S phase from α
factor arrest as shown in Fig 5. Cells were also shifted to 30°C during G1 arrest for 45 min to inactivate eco1-1 before
S phase. After release from α factor for 0, 60, and 120 min in nocodazole (15 µg/ml), plating efficiency was
determined on triplicated YPG (galactose) or YPD (glucose) plates after incubation at 23°C for 3 days. Standard
deviation of the mean was based on all available data.
Figure S4. Orc2 Reinduction at G2/M
Total cell extracts from strains cultured as in Fig 6B were subjected to Western blot analysis with anti-Orc2 antibody
to monitor the reinduction of orc2-1 protein.
Table S1. Yeast Strains Used in This Study
Strain
Genotype
Source
GA-180
GA-1629
GA-1680
GA-1778
GA-1780
GA-1782
GA-1783
GA-1877
GA-1878
GA-1906
GA-1908
GA-2029
GA-2030
GA-2146
GA-2147
GA-2169
GA-2170
GA-2203
GA-2204
GA-2216
GA-2218
GA-2243
GA-2244
GA-2245
GA-2250
GA-2251
GA-2273
GA-2295
GA-2386
GA-2387
GA-2825
GA-2826
GA-2887
GA-2288
GA-2289
GA-2290
GA-3006
GA-3590
GA-3591
GA-3831
GA-3832
GA-3848
GA-3849
MATa ade2-1 trp1-1 his3-11,15 ura3-1 leu2-3,112 can1-100
MATα ade2 his3 ura3 leu2 smc1::smc1-2-LEU2 brn1 ::BRN1-13Myc-KanMX6
GA-180 with orc2::His3MX6-UASGal-orc2-1
GA-1783 with orc2::His3MX6-UASGal-orc2-1 rad9::LEU2
GA-1783 with orc2::His3MX6-UASGal-orc2-1
GA-1783 with rad9::LEU2
GA-180 with URA3::GFP-TUB1
GA-180 with URA3::GPD-TK7x rad53::Rad53-13Myc-KanMX6
GA-1877 with orc2::His3MX6-UASGal-orc2-1
GA-180 with rad53::Rad53-13Myc-KanMX6
GA-1906 with orc2::His3MX6-UASGal-orc2-1
GA-1906 with mad2::URA3 rad9::LEU2 rad24::TRP1
GA-2029 with orc2::His3MX6-UASGal-orc2-1
GA-180 with TRP1::lac-op repeat HIS3::lac I-GFP
GA-2146 with orc2::His3MX6-UASGal-orc2-1
GA-180 with pds1::PDS1-3HA-URA3
GA-2169 with orc2::His3MX6-UASGal-orc2-1
GA-180 with scc1::SCC1-18Myc-TRP1
GA-180 with scc3::SCC3-18Myc-K.l.TRP
GA-2203 with orc2::His3MX6-UASGal-orc2-1
GA-2204 with orc2::His3MX6-UASGal-orc2-1
GA-180 with ARS1413::lac-op repeat -TRP1 HIS3::lac I-GFP
GA-2243 with orc2::His3MX6-UASGal-orc2-1
GA-2273 with orc2::His3MX6-UASGal-orc2-1
GA-2169 with chk1::His3MX6
GA-2169 with chk1::His3MX6 orc2::His3MX6-UASGal-orc2-1
GA-180 with ARS609::lac-op repeat -TRP1 HIS3::lac I-GFP
GA-2146 with cdc16
GA-180 with scc1::SCC1-GFP-KanMX6
GA-2386 with orc2::His3MX6-UASGal-orc2-1
GA-2146 with eco1-1
GA-2146 with eco1-1 orc2::His3MX6-UASGal-orc2-1
MATa ade2 his3 leu2 ura3 trp1 TRP1::lac-op repeat HIS3::lac I-GFP
GA-2287 with smc1::smc1-2-LEU2
GA-2287with orc2::His3MX6-UASGal-orc2-1
GA-2287 with smc1::smc1-2-LEU2 orc2::His3MX6-UASGal-orc2-1
GA-2295 with orc2::His3MX6-UASGal-orc2-1
GA180 with ura3::3xURA3 tetO112 UASURA3:20x ARS1-HIS3 leu2::TetR-GFP-LEU2 scc1::SCC1-18Myc-TRP1
GA180 with ura3::3xURA3 tetO112 UASURA3:20x mutARS1-HIS3 leu2::TetR-GFP-LEU2 scc1::SCC1-18Myc-TRP1
GA-3590 with sum1::SUM1-3FLAG-KanMX
GA-3591 with sum1::SUM1-3FLAG-KanMX
GA-3590 with brn1::BRN1-3HA-KanMX6
GA-3591 with brn1::BRN1-3HA-KanMX6
R. Rothstein (W303-1A)
D. Leroy
Shimada et al. (2002)
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K. Nasmyth (K6565)
K. Nasmyth (K7600)
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