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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 300l 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 10g/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 10g/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 10g/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 10g/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, 1M 6 Trichostatin A, 10mM NaButyrate, 1mM 3-Aminobenzamide, 1mM Sodium Orthovanadate, 50mM Sodium Fluoride, 50mM Disodium Glycerol-2-Phosphate, 1M 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 20l 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 50l SDS-PAGE sample buffer without reducing agent. After brief centrifugation, 40l of each supernatant was transferred into fresh Eppendorf tubes containing 0.4l 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 10g/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 2.5m 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 1g 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 10g/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