Supplementary Methods
Yeast transformation and plasmid shuffle
All of the 24 pol30 mutants were in Plasmids pBL-230-x (ARS, CEN TRP1 pol30x). Sixteen mutants were kindly provided by Peter Burgers 1,2. We made six additional
mutants based on information that the corresponding human PCNA mutants bound
weakly to the human p150 (CAC1) in vitro compared to wild type (K. Shibahara, Z.
Zhang, Morioka, H, T. Tsurimoto, and B. Stillman, unpublished) by procedures described
in the Instruction Manual for the QuikChangeTM Site-Directed Mutagenesis Kit
(Stratagen). Two mutants, pol30-203 and pol30-204 , were made based on information
that the corresponding Drosophila PCNA mutations suppressed position effect
varigation. Each of the PCNA mutants was introduced into yeast cells by plasmid shuffle.
To select for loss of the plasmid pBL-211 in yeast strains with the URA3 gene integrated
at the chromosome VIIL, individual colonies were tested by colony PCR using primers
pcna(sc)-3 and pcna(sc)-4 that recognized POL30 at its endogenous locus and on the
plasmid pBL-211, but not on the plasmid pBL230. To select for loss of the plasmid pBL211 in yeast strains without the URA3 gene on the chromosome VIIL, we replica-plated
yeast cells onto FOA plates, and FOA resistant colonies were picked.
C-terminal tagging of Cac1p and Cac2p
Cac1p and Cac2p were tagged at their C-termini with three HA epitopes and
thirteen Myc epitopes, respectively, using PCR modules described by Longtine et al. 3.
The PCR modules used were pFA6a-3HA-kanMX6 and pFA6a-13Myc-kanMX6,
respectively. The sequences of the all of the primers used in this study are listed in
Supplementary Table 3. The PCR modules were amplified by the Cloned pfu DNA
Polymerase (Stratagene). PCR products were purified, and used to transform the parental
yeast strain (W303-1, MATa) according to procedures as described 3. Clones in which
1
Cac1p or Cac2p were tagged at their C-termini were identified by colony PCR using one
primer (kan(r)-1) that annealed within the kan gene and another within the CAC1 gene
(cac1-f) or CAC2 gene (cac2-f) (Supplementary Table 3). The clones that gave rise to
PCR products with the expected size were further analyzed by Western Blotting using
monoclonal antibodies against the HA epitope (12CA5) or the Myc epitopye (9E10).
During subsequent crosses, the selection marker kanMX6 segregated 2:2, indicating that
only the kanMX6 gene was integrated into one locus in the genome. These two tags did
not affect their association with each other since monoclonal antibodies that recognized
the HA epitope specifically immunoprecipitated Cac2p-13Myc (data not shown).
Furthermore, these two tags did not affect the function of CAF-1 in silencing (data not
shown).
Production of antiserum against S. cerevisiae PCNA
The S. cerevisiae PCNA was expressed in E. coli and purified to homogeneity
according to procedures as described 4. The full-length protein was injected into rabbits,
and sera were tested. Antisera from the rabbit 871 recognized purified PCNA and reacted
with one protein from yeast whole cell extracts whose molecular weight matched that of
purified PCNA.
Cloning, expression and purification of GST-Cac1p
To clone pol30 mutants for in vitro transcription/translation, the entire coding
sequence of each of the mutants was amplified by PCR using primers pcna(sc)-6 and
pcna(sc)-7, and ligated into vector pT7Blue-2 (Novagen) to generate plasmids pZG10-13
as listed in Supplementary Table 4. Selected clones were sequenced to confirm no other
errors were introduced by PCR. Each of the PCNA mutants in pT7Blue-2 was subcloned
into the vector pET3c to generate plasmids pZG14-17. To clone GST-Cac1p, the entire
open reading frame of CAC1 was amplified by PCR using primers cac1(Eco RI) and
2
cac1(Xho I). The resulting PCR products were digested with EcoRI and Xho I and ligated
into vector pGEX-5X1 to generate plasmid pZG18. The coding region of CAC1 from
selected clones was sequenced to verify that no errors were introduced by PCR
amplification. All of the plasmids generated from this study were listed in the
Supplementary Table 4.
To express GST-Cac1p in BL-21(DE3), bacterial cells were grown in LB media
containing 50 g/ml Ampicillin at 24 ºC to OD600 of 0.5. They were then induced with
0.4 mM IPTG for five hours. After induction cells were harvested and frozen at –70 ºC.
Bacterial cells from 1 liter of LB culture were resuspended in 25 ml buffer A (25 mM
Tris, pH7.2, 10% glycerol, 0.05% NP40, 100 mM NaCl, 2 mM DTT) plus protease
inhibitors (1 mM PMSF, 10 g/ml leupeptin, 1 g/ml pepstatin A, 2 mM benzamidine,
10 g /ml aprotinin and 2 mM pefabloc), lysed by two passages through a French Press
and sonication. The resulting lysate was cleared by centrifugation, and loaded onto a 50
ml-Hydroxyapatite (Bio-Gel http Gel) column equilibrated with buffer A with 1 mM
PMSF. The column was washed with 100 ml of buffer A first, then 100 ml of buffer B
(300 mM Phosphate, pH 7.2, 10% glycerol, 0.05% NP-40). After washing, GST-Cac1p
was eluted with 100 ml of buffer C (600 mM Phosphate, pH 7.2, 10% glycerol, 0.05%
NP-40), incubated with 0.8 ml of Glutathione sepharose beads for at least 6 hours at 4 ºC.
The beads were then washed four times with 25 ml TBS each. Since GST-Cac1p was
very prone to proteolysis, we used the beads immediately for in vitro binding assay
described below. GST-REG was induced at 37 ºC for 2 hours, and purified by
glutathione sepharose beads. Protease inhibitors (1 mM PMSF, 2 mM pefabloc) were
added to buffer B, buffer C and TBS before use.
Southern Blot analysis of telomere length
3
Yeast DNA was isolated from vegetative cultures by a standard method 5. Two
g of DNA from each sample were digested with XhoI for 6 hours. The resulting
fragments were resolved on a 0.8% agarose gel, transferred overnight to a nitrocellulose
membrane, and hybridized with oligonucleotide (TG)20 labeled with 32P by T4
polynucleotide kinase.
Determination of sensitivity towards DNA damage agents
Yeast cells were grown to early logarithmic phase, and ten-fold serial dilutions
were made starting OD600 of 0.6. To determine sensitivity to UV induced DNA
damage, 5 l of each dilution were plated onto YPD plates. The plates were irradiated
with UV at a dosage of 0, 25, 50 and 100 J/m2, respectively, and incubated immediately
in dark at 30 ºC for three days. To test bleomycin sensitivity, 5 l of each dilution were
plated onto plates containing 0, 1 mU/ml, 3 mU/ml or 10 mU/ml bleomycin (Sigma).
Cells were grown at 30 ºC for three days before photography.
Supplementary references
1.
Ayyagari, R., Impellizzeri, K. J., Yoder, B. L., Gary, S. L. & Burgers, P. M. A
mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles
in DNA replication and DNA repair. Mol Cell Biol 15, 4420-4429 (1995).
2.
Eissenberg, J. C., Ayyagari, R., Gomes, X. V. & Burgers, P. M. Mutations in
yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA
polymerase delta and DNA polymerase epsilon. Mol Cell Biol 17, 6367-6378 (1997).
3.
Longtine, M. S. et al. Additional modules for versatile and economical PCRbased gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953-961
(1998).
4.
Fien, K. & Stillman, B. Identification of replication factor C from Saccharomyces
cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol
12, 155-163 (1992).
5.
Hoffman, C. S. & Winston, F. A ten-minute DNA preparation from yeast
efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57,
267-272 (1987).
4
6.
Kaufman, P. D., Kobayashi, R. & Stillman, B. Ultraviolet radiation sensitivity and
reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin
assembly factor-I. Genes Dev 11, 345-357 (1997).
7.
Game, J. C. & Kaufman, P. D. Role of Saccharomyces cerevisiae chromatin
assembly factor-I in repair of ultraviolet radiation damage in vivo. Genetics 151, 485-497
(1999).
5
Figure legend for the Supplementary Figures
Supplementary Figure 1. Conversion of “pink” colonies of the pol30-8 mutant to
sectoring colonies. Three pink and two sectoring colonies were picked from YPD plates
and assayed for the expression of the ADE2 gene at the HMR locus with time (X-axis)
and percentage of “pink” colonies in the whole population (Y-axis) was determined. Note
that sectored colonies remained predominantly pink after conversion.
Supplementary Figure 2.
Sensitivity towards DNA damage agents in pol30 and
pol30 cac1∆ mutants. A. Sensitivity towards UV irradiation. Ten-fold serial dilutions of
yeast cells with relevant genotypes identified at the left were dot-spotted onto YPD
plates, irradiated with UV at a dosage displayed at the top of the Supplementary Figure
2A. B, Sensitivity towards bleomycin. The same dilutions of yeast cells were dot-spotted
onto YPD plates containing the durg of bleomycin at concentrations exhibited at the top
of Supplementary Figure 2B. The photos were taken after growth in dark for three days
at 30 ºC.
Supplementary Figure 3.
Telomere length in wild type PCNA or mutants strains. A.
A schematic representation of yeast telomeres. About two-third of yeast telomeres have
both sub-telomeric repeats, Y’ and X. The Y’ element contains an unique XhoI site that is
about 1.3 kb from chromosome ends. B. Yeast genomic DNA from each of the strains
was digested with XhoI, resolved on a 0.8% agarose gel, transferred to a nitrocellulose
membrane, and hybridized with poly TG probe. Yeast strains used were WT, 1, 6, 8, 22,
41, 42, 45, 79 that corresponded to ZGY003-x (x=WT, 1, 6, 8, 22, 41, 42, 45, 79),
respectively, and cac1∆ (PKY106).
6
Supplementary Table 1
PCNA mutants affect silencing at telomere and HMR
Allele mutations
WT
1
2
6
8
9
13
79
14
16
81
18
22
41
42
45
46
200
201
202
203
204
205
206
207
wild type
EK3,5AA
EK5,7AA
DD41,42AA
RD61,63
ED104,105AA
DR109,110AA
LI126,128AA
KE127,129AA
EK143,146AA
DI156,158AA
DE187,189AA
DE256,257AA
Allele1+8
Allele 6+8
Allele 8+22
Allele 9+22
D97A
D122A
D256∆
P140L
I195E
DD97,122AA
DD97,256A∆
DD122,256A∆
FOA
Phenotype
hmr::ADE2
+++
+++
+++
++
+++
+++
+/+++
+++
+++
+++
+++
+
+
+++
+++
+++
+++
+++
+++
+++
+++
+++
P
P
ND
W
Sector, 5-10% LP
ND
ND
LP
ND
ND
ND
ND
P
Sector, 5-10% LP
W
Sector, 5-10% LP
ND
ND
ND
ND
ND
ND
ND
ND
ND
Each of the pol30 mutants with amino acid changes listed in the second column was
tested for telomeric silencing as described in Methods and reported as cell’s ability to
grow on FOA containing media. Cells with the wild type POL30 grew on FOA plates
quite efficiently (+++), suggesting that the URA3 gene was repressed. (+++) indicates
that pol30 mutants were indistinguishable in growth compared to the POL30 ; ++,
mutants exhibited minor growth difference; +, mutants could grow on FOA plates only at
the lowest dilution (about 5 x104 cells per spot); +/-, mutants barely grew on FOA plates;
-, mutants did not grow on FOA plates even at the lowest dilution. The growth was
scored after 3 days of incubation at 30 ºC. Selected pol30 mutants were also tested for
HMR repression using a qualitative colony color assay: P, pink; LP, light pink, W, white;
7
Sector, LP/W sectored colony. ND, not determined. The pol30 mutants showing
reduction in silencing were highlighted.
8
Supplementary Table 2
Yeast strains used
Strain
Number
Reference
PKY090
PKY106
PKY741
RS1295
ZGY001
ZGY002
ZGY003
ZGY003-x
ZGY004
ZGY004-x
ZGY005
ZGY005-x
ZGY006
ZGY006-x
ZGY007
ZGY007-x
ZGY008
ZGY008-x
ZGY009
ZGY101
ZGY110
Kaufman et al. 6
MATa URA3-VIIL
same as above
MATa URA3-VIIL cac1∆::Leu2
Game & Kaufman 7 MATa pol30∆::hisG-URA3-hisG+pBL230(POL30)
Rolf Sternglanz
MATa, hmr::ADE2
this study
MATa pol30∆+pBL230(POL30)
this study
MATa pol30∆+ [pBL-211(POL30 URA3)]
this study
MAT pol30∆ URA3-VIIL [pBL-211(POL30 URA3)]
this study
ZGY003+[pBL230-x TRP1]-[pBL-211]
this study
ZGY002 CAC1-3HA CAC2-13MYC
this study
ZGY004 +[pBL230-x TRP1]-[pBL-211]
this study
ZGY002 hmr::ADE2
this study
ZGY005 +[pBL230-x TRP1]-[pBL-211]
this study
ZGY002 CAC1-3HA
this study
ZGY006 +[pBL230-x TRP1]-[pBL-211]
this study
ZGY003 cac1∆::LEU2
this study
ZGY007 +[pBL230-x TRP1]-[pBL-211]
this study
ZGY005 cac1∆::LEU2
this study
ZGY008 +[pBL230-x TRP1]-[pBL-211]
this study
cac1∆::LEU2 hmr::ADE2
this study
MATa cac2::CAC2 13myc -kanMX6
this study
MATa cac1∆::LEU2 cac2::CAC2 13myc -kanMX6
Genotype
All of the yeast strains were derivative of W303-1 (leu2-3,112 ura3-1 his3-11,15 trp1-1 ade2-1
can1-100). X= wild type PCNA or mutant alleles listed in Supplementary Table 1.
9
Supplementary Table 3 Sequences of PCR primers used in this Study
Name
pcna(sc)-3
pcna(sc)-4
pcna(sc)-6
pcna(sc)-7
cac1(EcoRI)
cac1(XhoI)
cac1(f)
cac2(f)
kan(r)-1
cac1(tag)-f
cac1(tag)-r
cac2(tag)-f
cac2(tag)-r
DNA sequence
5’ GTAGCTGACGGTGATATCGG 3’
5’ GTTGGATGTGGAAACCCTGAATAC 3’
5’ CATATGTTAGAAGCAAAATTTGAAGAAGC 3’
5’ TTATTCTTCGTCATTAAATTTAGGAGC 3’
5’ AAAAGAATTCATGGAGCAACATCTCAAATCAATTC 3’
5’ AAAACTCGAGTTACAAAGACGGGGTTGGCATATTTGC
5’ CCCATTGATACCAACAATATGCC 3’
5’ ACTGAACAGCGCTGGTGGCG 3’
5’CGTATGGGTAAAAGATGTTAATTAAC 3’
5’ CGCAAATGGGTAATCAAAGACGCACAAAACTGGGAGA
ATCTTCGAGCCAATGCAAATATGCCAACCCCGTCTTTGC
GGATCCCCGGGTTAATTAA 3’
5’ TTATTCAAAGTCCAAAAGGCTTGACTGTCGATCGTAGT
GTTGTCGCCTTTTTCATGTATACCAATAAATAATCAGGAA
TTCGAGCTCGTTTAAAC 3’
5’ ATCCCCTGCAATAGTAGCGATAGTAAAAAGAGGCGC
ATACATCCTACGCCAGTCGATTTGCGGATCCCCGGGTTA
ATTAA 3’
5’ ATATCTTCGCTGGCAAACTAGATTAGGCATTCTTATGT
ACCGCATTAAATATATTAAAAAGAATTCGAGCTCGTTTA
AAC 3’
10
Supplementary Table 4
Plasmids constructed in this study
Name
Gene or
mutant allele
Parental Plasmid
pol30-200
pol30-201
pol30-202
pol30-203
pol30-204
pol30-205
pol30-206
pol30-207
pol30-0
pol30-6
pol30-8
pol30-42
pol30-79
pol30-6
pol30-8
pol30-42
pol30-79
CAC1
pBL-230
pBL-230
pBL-230
pBL-230
pBL-230
pBL-230
pBL-230
pBL-230
pT7 Blue-2
pT7 Blue-2
pT7 Blue-2
pT7 Blue-2
pT7 Blue-2
pET3c
pET3c
pET3c
pET3c
pGEX-5x-1
pZG1
pZG2
pZG3
pZG4
pZG5
pZG6
pZG7
pZG8
pZG9
pZG10
pZG11
pZG12
pZG13
pZG14
pZG15
pZG16
pZG-17
pZG-18
11