Download Cell cycle analysis

Supplementary Methods
Yeast strains
A list of yeast strains used for this work is supplied as supplementary Table 1. Unless
stated otherwise, the parental strain that we used was HMY57 (hht2-hhf2::kanMX3) in
which YIplac201 plasmids carrying either wild-type HHT1 or hht1 mutations (see
below) were integrated at the HHT1 locus. The wild-type HHT1-HHF1 locus was subcloned in the high-copy plasmid YEplac181 for overexpression studies or in the lowcopy centromeric plasmid YCplac22 to create histone H3 point mutations. Strains that
express hht1 mutants as the only source of histone H3 and H4 were generated by
transforming the YCplac22 constructs into the plasmid shuffle strain DY5733 (hht1hhf1::LEU2 hht2-hhf2::KANMX3 [YCp50 HHT2-HHF2 URA3]) and the YCp50
plasmid encoding wild-type H3 was selected against on 5-fluoro-orotic acid (5-FOA)
plates. For Figure 4, YIplac201-based plasmids encoding HHT1-HHF1, hht1 K56QHHF1 or hht1 K56R-HHF1 were integrated at the TRP1 locus into DY5733 and the
YCp50 plasmid encoding wild-type H3 was selected against on 5-FOA (strains
HMY139, HMY140 and HMY152). An integration vector to tag the C-terminus of
1
Top1 with 3 FLAG epitopes was created by sub-cloning a fragment encoding the Cterminal 531bp of the TOP1 ORF, without the stop codon, into the NotI and BamHI
sites of the pRS305-3FLAG vector. This construct was linearised with SpeI for
integration at the TOP1 locus. For gene disruption, we used a polymerase chain reaction
(PCR) method1. Mutations and gene disruptions were confirmed by PCR, Southern
blotting and DNA sequencing.
Yeast strain HMY110 was used for purification and mass spectrometry of epitopetagged histone H3 (Figure 1a). Yeast strains HMY57 (wild-type) and HMY160 (hht1
K56R) were used in Figure 1c-e and 1h. Strains YKB2 (cdc7-4) and YDL6 (CAC13FLAG cdc7-4) were employed in Figure 1f. Strain HMY73 (pGAL1-HHT1) was used
for Figure 1g. Strain HMY133 (wild-type), HMY134 (hht1 K56A), HMY135 (hht1
K56Q), HMY136 (hht1 K56R) and YJT75 (rad53 sml1) were used in Figure 2a.
Strains HMY133 (wild-type), HMY136 (hht1 K56R), CY1532 (hhf1-10), CY184 (wildtype) and Y01177 (hta1 S129A hta2 S129A) were used for Figure 2b. Strains derived
from DY5733 by plasmid shuffling and containing high-copy YEplac181 plasmids
2
encoding either wild-type HHT1 or the hht1 K56R allele were used for Figure 2c.
Strains HMY57 (wild-type), HMY140 (hht1 K56R), HMY142 (rad52) and HMY145
(hht1 K56R rad52) were used for Figure 2d. Strains HMY57 (wild-type) and
HMY198 (rad9) were used for Figures 3a and 3b, respectively. Strains YAV149
(TOP1-3FLAG wild-type) and YAV150 (TOP1-3FLAG rad9) were used for Figure
3d. Strains HMY152 (wild-type), HMY139 (hht1 K56Q) and HMY140 (hht1 K56R)
were used for Figures 4b and 4c. The strains used for the Supplementary Figures are
indicated in each legend.
Tagged histone H3 purification
Cells expressing FLAG-His10-HHT1 (FH-H3) as the only source of histone H3 (2.4
x1010 cells) were treated with 20ml of 0.1M NaOH and incubated for 5min at room
temperature2. After centrifugation at 6000rpm for 5min, the cell pellet was resuspended
in 10ml of Lysis buffer (60mM Tris-HCl pH 6.8, 5% Glycerol, 2% SDS, 4% 2mercaptoethanol) and boiled for 5min. After centrifugation at 6000rpm for 5min, the
cell lysate was recovered, diluted four-fold in Dilution buffer (10mM Tris, 100mM
3
NaPhosphate pH 8.0, 8M Urea) and mixed with 1ml of Nickel-NTA agarose beads
(Qiagen). After incubation for 2h at 4°C, the beads were washed 3 times with dilution
buffer and then three times with Wash buffer (10mM NaPhosphate pH 8.0, 500mM
NaCl, 0.5% NP-40, 10mM Imidazole). FH-H3 was eluted from the Nickel-NTA beads
with Elution buffer (10mM NaPhosphate pH 8.0, 500mM NaCl, 0.5% NP-40, 400mM
Imidazole) and then immunoprecipitated with 300l of FLAG M2 antibody beads. After
washing with Wash buffer, FH-H3 was eluted from the beads with Dilution buffer.
Quantitation of the degree of histone H3 K56 acetylation
To determine the fraction of histone H3 acetylated at K56, untagged histones were
purified from asynchronous cells as described3. Histone H3 was resolved in an SDS15% polyacrylamide gel and digested with endoproteinase Arg-C. The digest was
evaporated in a microcentrifuge tube and treated with 5% d6 (deuterated)-acetic
anhydride, 5% triethylamine in acetonitrile for 1 hour at room temperature. The
reaction was dried in a vacuum centrifuge and the residue redissolved in 1% formic acid
for analysis by mass spectrometry.
4
Fractionation of soluble protein and chromatin and detection of histones bound to
Cac1-3FLAG
These procedures were previously described4.
DNA damage sensitivity assays
Strains were grown overnight at 30°C in either rich YPD medium or minimal medium
for plasmid selection. Cells were diluted to 6x106 cells/ml, and grown for 3h. Cultures
were diluted to equivalent cell densities, and ten-fold serial dilutions were plated onto
media containing the indicated DNA damaging agents. Plasmid end-joining assays were
performed as previously described6.
Cell cycle analysis
Cells in exponential phase were arrested in G1 using 10g/ml -factor for 2-3h,
washed, and released into the cell cycle in fresh YPD medium at 25°C either in the
absence or presence of 10g/ml CPT. Aliquots (1ml) were taken at 15- or 30-min
5
intervals for Western blots2 and DNA content determination by FACS analysis.
Additionally, 200 cells of each time point were analysed for budding by microscopy.
Supercoiling and micrococcal nuclease digestion assays
These assays were performed as described7,8.
Affinity purification of oligonucleosomes covalently attached to Top1-FLAG3
1-litre cultures at 1x107 cells/ml were treated with 10g/ml -factor for 2h 15min at
30°C to synchronise cells in G1. The cells were harvested (5min at 3000rpm at 25°C),
resuspended in 1 litre of warm YPD medium (30°C) containing 10g/ml camptothecin.
After completion of one round of DNA replication in the presence of camptothecin
(50min at 30°C), the cultures were chilled on ice and the cells were harvested by
centrifugation (5min at 3000rpm and 4°C). The cells were washed twice with ice-cold
water and the cell pellets frozen on dry ice and stored at -80°C. Cell pellets were
resuspended in 15ml of lysis buffer (20mM Hepes/NaOH pH 7.5, 300mM Sodium
Acetate pH 7.5, 10% Glycerol, 0.1% Tween-20, 10mM 2-Mercaptoethanol, 1M
6
Trichostatin A, 10mM NaButyrate, 1mM 3-Aminobenzamide, 1mM Sodium
Orthovanadate, 50mM Sodium Fluoride, 50mM Disodium Glycerol-2-Phosphate, 1M
MG132, 1X EDTA-Free Roche Protease Inhibitor Cocktail) and the suspension was
added dropwise into 50-ml Falcon tubes containing liquid nitrogen to create frozen
beads of yeast cell suspension. These beads were subjected to mechanical disruption
under liquid nitrogen in a Spex freezer mill (4 disruption cycles of 2min each at 15
pulses/sec with 2min cooling intervals). After thawing out on ice (1-2h), chromatin was
fragmented with the large probe of a Branson sonifier (7 cycles of 15sec each at 40%
output power with 2min cooling intervals on ice). DNA extracted after the sonication
step and analysed in a 1.5% agarose gel consisted of a uniform smear extending from
~200bp (mono-nucleosome) up to ~1.5kb (10-nucleosome long fragments). Insoluble
aggregates were removed by centrifugation (20min, 16000rpm, 4°C, Beckman SS34).
The soluble fraction was incubated overnight (~12h) with 20l of agarose beads coated
with the FLAG M2 mouse monoclonal antibody (Sigma). After centrifugation (2min,
2500rpm, 4°C, Beckman R6K), the supernatant was discarded and the beads were
washed four times with 1.5ml of freshly prepared lysis buffer. The beads were boiled
7
for 2min in 50l SDS-PAGE sample buffer without reducing agent. After brief
centrifugation, 40l of each supernatant was transferred into fresh Eppendorf tubes
containing 0.4l of 14.3M 2-mercaptoethanol (143mM final) and re-boiled for 2min
under reducing conditions. The samples were resolved in SDS-15% polyacrylamide
gels, which were transferred onto nitrocellulose in 10mM CAPS (NaOH) pH 11, 20%
methanol for 2 hours at 70V. The upper portion of the blots was probed with FLAG M2
antibodies to detect Top1-3FLAG and the lower portion was used to detect the
modifications of histones co-precipitated with Top1.
8
Supplementary Table 1 Strains used in this study
Name
CY1532
CY184
HMY57
HMY73
HMY110
HMY133
HMY134
HMY135
HMY136
HMY139
HMY140
HMY142
HMY145
HMY146
HMY152
HMY160
HMY165
HMY198
9
Genotype
MATa lys2-201 leu2-3,112 HHT1 hhf1-10 (hht2-hhf2)
MAT ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1
rDNA::ADE3
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht2-hhf2::kanMX3
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht2-hhf2::kanMX3 hht1::TRP1
ura3:: PGAL1-HHT1::URA3
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
[YCp22 FLAG-His10-HHT1 HHF1 TRP1]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
[YCp22 HHT1 HHF1 TRP1]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
[YCp22 hht1 K56A HHF1 TRP1]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
[YCp22 hht1 K56Q HHF1 TRP1]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
[YCp22 hht1 K56R HHF1 TRP1]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
trp1:: hht1 K56Q-HHF1:: TRP1
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
trp1::hht1 K56R-HHF1:: TRP1
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hhtf2-hht2::kanMX3
rad52::his5+(S. pombe)
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1 K56R::TRP1 hht2-hhf2::kanMX3
rad52::his5+(S. pombe)
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht2-hhf2::kanMX3
hdf1(yku70)::his5+(S. pombe)
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
trp1:: HHT1-HHF1:: TRP1
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1 K56R::TRP1 hht2-hhf2::kanMX3
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht2-hhf2::kanMX3 mre11::his5+ (S. pombe)
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht2-hhf2::kanMX3 rad9::his5+(S. pombe)
Source
Bird, et al. (2002)9
Bird, et al. (2002)9
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
HMY201
U953-61A
Y01177
YKB2
YDL6
DY5733
YJT75
YAV149
YAV150
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht2-hhf2::kanMX3 cdc7-4
[YEp195 PGAL1 HTH-HHT2 URA3]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 mec1::TRP1 sml1::HIS3
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hta1 S129A hta2 S129A
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 cdc7-4
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 cdc7-4 CAC1-3FLAG::TRP1
This study
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 hht1-hhf1::LEU2 hht2-hhf2::kanMX3
[YCp50 HHT2-HHF2 URA3]
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 sml1::URA3 rad53::LEU2
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 TOP1-FLAG3::LEU2
MATa trp1-1 ura3-1 his3-11,15 leu2-3,112 ade2-1
can1-100 rad9::URA3 TOP1-FLAG3::LEU2
Wittschieben
et al. (2000)12
Zhao et al.
(1998)10
Redon et al.
(2003)5
Bousset and
Diffley (1998)11
This study
Tercero and
Diffley (2001)13
This study
This study
All strains were derived from W303 MATa14, except for the CY185 and CY1532 strains.
10
Supplementary References
1. Longtine, M. S. et al. Additional modules for versatile and economical PCR based
gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953-961
(1998).
2. Kushnirov, V.V. Rapid and reliable protein extraction from yeast. Yeast 16, 857-860
(2000).
3. Poveda, A. et al. Hif1 is a component of yeast histone acetyltransferase B, a complex
mainly localized in the nucleus. J. Biol. Chem. 279, 16033-16043 (2004).
4. Gunjan, A. & Verreault, A. A Rad53 kinase-dependent surveillance mechanism that
regulates histone protein levels in S. cerevisiae. Cell 115, 537-549 (2003).
5. Redon, C. et al. Yeast histone 2A serine 129 is essential for the efficient repair of
checkpoint-blind DNA damage. EMBO Rep. 4, 678-684 (2003).
6. Boulton, S.J. & Jackson, S.P. Saccharomyces cerevisiae Ku70 potentiates illegitimate
DNA double-strand break repair and serves as a barrier to error-prone DNA repair
pathways. EMBO J. 15, 5093-5103 (1996).
7. Downs, J.A., Lowndes, N.F. & Jackson, S.P. A role for Saccharomyces cerevisiae
histone H2A in DNA repair. Nature 408, 1001-1004 (2000).
8. Wechser, M.A., Kladde, M.P., Alfieri, J.A. & Peterson, C.L. Effects of Sin- versions
of histone H4 on yeast chromatin structure and function. EMBO J. 16, 2086-2095
(1997).
9. Bird, A.W. et al. Acetylation of histone H4 by Esa1 is required for DNA doublestrand break repair. Nature 419, 411-415 (2002).
10. Zhao, X., Muller, E.G.D. & Rothstein, R. A suppressor of two essential checkpoint
genes identifies a novel protein that negatively affects dNTP pools. Mol. Cell 2, 329340 (1998).
11. Bousset, K. & Diffley, J.F. The Cdc7 protein kinase is required for origin firing
during S phase. Genes Dev. 12, 480-490 (1998).
12. Wittschieben, B.O., Fellows, J., Du, W., Stillman, D.J. & Svejstrup, J.Q.
Overlapping roles for the histone acetyltransferase activities of SAGA and
elongator in vivo. EMBO J. 19, 3060-3068 (2000).
11
13. Tercero, J.A. & Diffley, J.F. Regulation of DNA replication fork progression
through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412, 553-537
(2001).
14. Thomas, B.J. & Rothstein, R. Elevated recombination rates in transcriptionally
active DNA. Cell 56, 619-630 (1989).
12
Supplementary Figure S1 Peptides derived from endoproteinase Arg-C digestion of
Flag-His10-H3 were analyzed on a nano-LC pepmap reversed-phase chromatography
C18 column (75µm x 150mm, LC-Packings) connected to a Q star tandem mass
spectrometer (Applied Biosystems/MDS Sciex). Two doubly charged parent ions with
m/z ratio of 617.83 and 638.85 were identified for peptide 54-63 (FQKSTELLIR) of
histone H3. The identities of these peptides were confirmed by collision-induced
fragmentation as unacetylated (m/z ratio = 617.83) and K56-acetylated (m/z ratio =
638.85). Due to the retention of the K56 positive charge, synthetic peptides that were
unmodified or K56-trimethylated eluted from the reversed-phase column similarly to
the unmodified parent ion peptide, whereas a K56-acetylated synthetic peptide lacking
the charge eluted much later at the position of the modified parent ion peptide.
Supplementary Figure S2 K56 acetylation predominantly occurs in S-phase cells. a,
Western blots of whole-cell extracts derived from wild-type cells (HMY57) in log phase
(Log) or cells arrested in G1 phase with -factor (), S phase with hydroxyurea (HU)
or released from -factor arrest into a nocodazole arrest ( to Noc). cdc7-4 mutant
13
cells (YKB2) were also released from a G1 arrest at the restrictive temperature of 38°C
( to cdc7-4). b, cdc7-4 cells containing a high-copy plasmid for galactose-inducible
expression of epitope-tagged histone H3 (HMY201) were either held in G1 or released
at the restrictive temperature for the cdc7 mutation (1h at 38°C). Expression of tagged
histone H3 was induced by the addition of galactose (Gal).
Supplementary Figure S3 ~20% of total histone H3 is K56-acetylated in asynchronous
cells. Non-tagged histone H3 purified from wild-type cells (W303 MATa) was digested
with endoproteinase Arg-C and the resulting peptides were acetylated in vitro with
deuterated acetic anhydride to render the original unmodified and K56-acetylated
peptides chemically equivalent before analysis by mass spectrometry. Both species are
acetylated with deuterium on their -amino groups, but they differ in mass because the
peptide that was K56-acetylated in vivo cannot be modified with a deuterated acetyl
group on the -amino group of K56 in vitro. The peaks are separated by 0.5 m/z ratio
units because the parent ions are doubly charged. The total surface area under the peaks
derived from each species was calculated to estimate the ratio of unmodified to K56-
14
acetylated histone H3.
Supplementary Figure S4 Mutations that affect histone H3 K56 confer sensitivity to
methyl methane sulphonate (MMS), hydroxyurea (HU) and camptothecin (CPT). a, 10fold serial dilutions of wild-type (HMY 133) and isogenic hht1 K56A (HMY 134),
K56Q (HMY 135), K56R (HMY 136) and rad53 sml1 (YJT 75) mutant strains were
analysed for colony formation on plates containing MMS and HU. b, Survival assays
were performed with wild-type (HMY57) and hht1 K56R mutant cells (HMY160)
released from G1 arrest in rich medium containing 10g/ml CPT. Cell survival at time
zero was taken as 100%. Cell cycle progression was monitored by measuring DNA
content by FACS.
Supplementary Figure S5 Histone H3 K56R mutant cells do not exhibit premature
mitotic spindle elongation in response to replication arrest. Wild-type (HMY 57), hht1
K56R (HMY 160) and rad53 sml1 (YJT 75) mutant strains were arrested in G1 and
then released in rich medium containing 200mM HU to inhibit DNA replication. After 3
15
hours at 24°C, cells were fixed with formaldehyde, stained with tubulin antibodies
(Serotec) and analysed by fluorescence microscopy to measure spindle elongation as a
marker of anaphase entry. Cells containing microtubule spindles longer than 2.5m
were scored as anaphase cells. 100 cells of each strain were counted.
Supplementary Figure S6 The DNA damage sensitivity of hht1 K56R mutant cells
cannot be accounted for simply by defects in Non-Homologous DNA End-Joining
(NHEJ). a, 10-fold serial dilutions of wild-type (HMY 57), hht1 K56R (HMY 160) and
yku70 (HMY 146) mutant strains were analysed for colony formation on plates
containing either MMS or CPT. b, The same strains as in a were assayed for NHEJ by
transforming cells with 1g of either EcoRI-linearised or circular YCp33 plasmid as
control6. The % repair was calculated as the ratio of the number of colonies obtained
with the EcoRI-linearised versus the uncut plasmid. The % repair was set to 100% for
wild-type (wt) cells.
Supplementary Figure S7 Histone H3 K56 contributes to MMS survival in a Rad52-
16
independent manner. Wild-type (HMY57), hht1 K56R (HMY160), rad52 (HMY142)
and rad52 hht1 K56R (HMY145) mutant cells were synchronised in G1 and released
into the cell cycle in the presence of 0.033% MMS. The fraction of viable cells was
determined as a function of time and survival at time zero was taken as 100%.
Supplementary Figure S8 Histone H3 K56 acetylation is not required for histone
modifications induced by ionising radiation. a, High levels of histone H3 K56
acetylation are not necessary for H2A phosphorylation in response to ionising radiation.
Asynchronous (Log) wild-type cells (HMY57) or cells arrested in G1 with -factor or
G2/M phase with nocodazole were exposed to 150Gy of -radiation from a Cs137
source. b, Asynchronous (Log) wild-type (HMY57) or hht1 K56R cells (HMY160)
were exposed to 150Gy of -radiation. Western blots of whole-cell lysates were probed
with antibodies that recognise histone H3 acetylated at K56, total histone H3, H2A
phosphorylation and histone H4 acetylated at K8. c, Asynchronous wild-type (HMY57),
hht1 K56R (HMY160) and rad52 (HMY142) mutant cells were exposed to 400Gy of radiation and cell survival measured by a colony formation assay.
17
Supplementary Figure S9 Mec1 but not Mre11 is needed to maintain histone H3 K56
acetylation in G2 cells that contain CPT-induced DSBs. a-b, mre11 (HMY165) and
mec1 sml1 cells (U953-61A) were released from G1 arrest in the absence or presence
of 10g/ml CPT at 25°C. Western blots of whole-cell extracts were probed for histone
H3 K56 acetylation, total H3 or H2A phosphorylation. Cell cycle progression was
monitored by assessing the budding index by microscopy and the cellular DNA content
by FACS.
18