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Supplementary Material Involvement of a chromatin remodeling complex in damage tolerance during DNA replication Karina B. Falbo1#, Constance Alabert2#, Yuki Katou3#, Su Wu4, Junhong Han5, Tammy Wehr1, Jing Xiao1, Xiangwei He6, Zhiguo Zhang5, Yang Shi4, Katsu Shirahige3, Philippe Pasero2 and Xuetong Shen1*

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Supplementary Figure 1. Example of Ino80 binding to ARSs during S phase. ChIP-Chip analysis was performed with an anti-FLAG antibody using a FLAG-INO80 strain as described in Fig. 1a and methods1. Data from Chromosome VI are shown as a representative example. Vertical bars represent the binding ratio of proteins in each locus and the horizontal axis shows kilo base units. The scale of the vertical axis is the signal log ratio1. Squares indicate early ARSs and rectangles indicate late ARSs. Horizontal blue bars represents genes.

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Supplementary Figure 2. INO80 is required to start MMS stalled forks. (a) γ-H2AX detection by

western blot. Early log-phase culture of wildtype (WT) and ino80 (∆) mutant cells were arrested in G1 and treated with 0.02% MMS for half an hour before release into the S phase. Cultures were then washed and divided in two. One half was released into the S phase in media with 0.02 % MMS. The other half was re-suspended in media with MMS and α-factor to keep cells in G1 block. Samples were

taken from both cultures simultaneously and analyzed by western blotting using an anti-γ-H2AX

antibody (ab15083 Abcam). Clb2 is a marker of cell cycle progression (sc-9071 Santa Cruz Biotechnology). Actin is a loading control. (b-d ) PFGE analysis of the arp8 mutant. Fork recovery after an acute exposure to MMS was analyzed in wildtype (WT, PP633) and arp8 (PP829) cells. Cultures were released from G1 arrest into the S phase in the presence or absence of 0.033% MMS. Cells exposed to MMS were washed after 60 minutes with 2.5% sodium thiosulfate to inactivate MMS. After MMS inactivation cells were re-suspended in fresh medium and samples were collected at the indicated times for further analysis (see Fig. 2c). (b) Flow cytometry analysis of DNA content in

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a normal S phase and during recovery after MMS treatment. Time 0 of release from MMS was taken 60 minutes after release from α-factor in the presence of MMS. (c) Budding index of wildtype and

arp8 cells released from G1 in the absence of MMS (top). The viability of wildtype and arp8 cells after an acute exposure to 0.033% MMS is indicated (bottom). (d) The pulsed field gel from Fig. 2c was transferred to a nylon membrane and chromosomes III, XII and XIV were detected by Southern blot2. Open circles represents arp8 mutant. (e) DNA combing analysis of the arp8 mutant (see also Fig. 2 e-g). Fork recovery was analyzed by DNA combing after and acute exposure to MMS in wildtype and arp8 cells3. Images of DNA combing are shown for samples taken at 0, 90 and 130 minutes after release from MMS. See Methods for experimental details. Green: BrdU. Red: ssDNA. Asterisks: stalled forks. Bar: 50 kb.

Supplementary Figure 3

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Supplementary Figure 3. INO80 is involved in the DNA damage tolerance pathway. (a) PCNA ubiquitination in asynchronous cultures of wildtype (WT) or K164R, a PCNA point mutant strain that cannot be ubiquitinated. G1 synchronized cultures were released into the S phase in media containing 0.02% MMS for an hour and PCNA ubiquitination was examined using the same protocol4 as in Figure 3ab. (b) Histone H3 K56 acetylation indicates normal replication in the ino80 mutant. K56 acetylation on histone H3 was monitored in cells progressing through the S phase using an antibody that specifically recognizes the acetylated form of Lys56 in histone H3 (Upstate, cat. # 07-677). Histone H3 expression was included as a control (ab1791 from Abcam). Clb2 was included as a marker of cell cycle progression (a slight increase of H3K56 acetylation levels is sometimes seen). (c) PCNA modification in MMS treated asynchronous cultures. Early-log phase wildtype (WT) and ino80 cultures were incubated with 0.02% MMS for up to 150 minutes. An Anti-PCNA antibody was used to detect PCNA and its modified forms from whole cell extracts. rad5 and rad6 are negative controls. Actin is a loading control. SUMO is the sumoylated form of PCNA and Ub2 is the di-ubiquitinated form

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of PCNA. Mono-ubiquitinated PCNA is not readily detected using this assay as described5. (d) PCNA modification in a strain with a point mutation (K737A) that abolishes the ATPase activity of Ino80 (ino80pGAT). ino80pWT is a ino80 null mutant with a wildtype INO80 plasmid. Experimental protocol as in d. (e) Replication progresses synchronously in both wildtype (WT) and the ino80 mutant. FACS analysis of samples taken from Rad18 ChIP experiment (Fig 3c) is used to monitor the progression of replication. Wild type (WT) and ino80 cells Flag-tagged at the RAD18 locus, were synchronized in G1, treated with 0.02% MMS in G1, then released into the S phase in media containing 100 mM HU and 0.02% MMS. Despite a slowdown of replication in ino80 mutant due to defects in DNA damage tolerance, S-phase entry and the synchrony of replication are normal in the ino80 mutant. Data are analyzed from three independent experiments.

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Supplementary Figure 4. INO80 is required for Rad51-dependent recombination intermediates formation after MMS treatment. (a) Kinetic analysis of X-spike intermediates formation in the arp8 mutant. Analysis of replication intermediates after MMS treatment at the early origin ARS305 by 2D gel electrophoresis2. Samples were collected 60, 105 and 130 after release into S phase with 0.1% MMS from wildtype (WT, PP633), arp8 (PP829) and sgs1 (PP118) cultures (see Fig. 4a-c and experimental procedures for details). Arrowhead: X-spike. (b) Rad51 recruitment to multiple ARSs. ChIP analysis of Rad51 recruitment to ARSs in S phase synchronized cells. Wildtype (WT) and ino80 cells were synchronized in G1, treated with 0.02% MMS in G1, then released into the S phase in media containing 100 mM HU and 0.02% MMS. Rad51 was immunoprecipitated with an anti-Rad51 specific antibody. Primers used correspond to ARS501, ARS601, ARS1212 and a control non-ARS

sequence in chromosome VI. Data are analyzed from three independent experiments. For further details see Fig. 3c,d and methods.

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Supplementary Figure 5. Model depicting the involvement of INO80 in DNA damage tolerance pathways. This model provides details to complement the model in Fig. 4d. In this model, after replication fork encounters obstructions caused by DNA damage (black triangles), the INO80 chromatin remodeling complex is recruited to the blocked replication fork. INO80 chromatin remodeling activity remodels the chromatin environment to facilitate the recruitment of factors from both the RAD6 and RAD51 pathways, such as Rad18 and Rad51. These initiating factors activate subsequent pathways to resolve the DNA damage-induced blockage at the replication fork, allowing DNA replication to resume and complete DNA replication without generating genome instability.

Supplementary Figure 6. Ino80 function at the replication fork is distinct from its function at DSB repair. (a-c) Arp8 but not Nhp10 is required for efficient replication fork restart after MMS treatment.

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Wildtype (WT, PP633) arp8 (PP829) and nhp10 (PP) cells were synchronized in G1 with α-factor and

released into the S phase for 60 minutes in the presence of MMS. Samples for PFGE were collected

at the indicated times after release from MMS. (a) PFGE analysis of S-phase completion after MMS. (b) Quantitation of chromosome mobility calculated for six representative chromosomes. (c) Budding index for each strain during normal S phase. (d) Replication fork restart by uSCE after MMS treatment is less efficient in arp8 mutant compared to wildtype. Asynchronous wildtype (WT, PP915) rtt101 (PP916) nhp10 (PP975) and arp8 (PP977) cells were plated on –HIS and YPAD plates immediately or after 45 min incubation in 0.02% MMS. Relative units of uSCE efficiency are estimated as described2 in methods.

Supplementary Table 1 INO80-upregulated genes Biological process INO80 Control (ino80Δ) HUG1 not yet annotated PHO12 biological_process unknown PHO11 biological_process unknown SPL2 cell cycle PHO84 phosphate transport DIA1 pseudohyphal growth HOR7 stress response MRH1 biological_process unknown ORF:YOR385W biological_process unknown YGP1 stress response PHO5 phosphate metabolism TIR3 biological_process unknown CIT2 glutamate biosynthesis WTM1 meiosis VPS73 biological_process unknown HXT1 hexose transport ASP3-4 nitrogen starvation response ASP3-1 nitrogen starvation response ASP3-2 nitrogen starvation response ASP3-3 nitrogen starvation response CMK2 protein amino acid phosphorylation ORF:YDR476C biological_process unknown PHO3 thiamin transport VTC1 vacuole fusion (non-autophagic) CAR1 not yet annotated HOR2 response to osmotic stress ORF:YJL144W biological_process unknown ERV1 iron homeostasis

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GPD1 intracellular accumulation of glycerol ORF:YDL241W biological_process unknown PRM7 mating ORF:YDL038C biological_process unknown ORF:YLR194C biological_process unknown ORF:YIL082W-A biological_process unknown TIR1 cell wall organization and biogenesis GPM2 gluconeogenesis CPR2 stress response ORF:YDR391C biological_process unknown ODC2 transport NMD5 biological_process unknown ORF:YNL058C biological_process unknown ERG1 ergosterol biosynthesis PHM6 biological_process unknown LYS9 not yet annotated HXT3 hexose transport ORF:YMR173W-A biological_process unknown GTT1 glutathione metabolism SPI1 biological_process unknown ARD2 biological_process unknown

INO80 down-regulated genes Biological process TRP1 Control (TRP1+) FIT1 Biological process unknown ADH2 Ethanol metabolism HSP12 Response to oxidative stress HMX1 Biological process unknown CYC1 Oxidative phosphorylation NEC103 Not yet annotated TSA2 Regulation of redox homeostasis SRL1 Nucleic acid metabolism YOR248W Biological process unknown SNZ3 Vitamin B6 metabolism

Supplementary Table 1. Transcriptional analysis of the ino80 mutant using microarrays. An ino80 culture was synchronized in G1 and released into the S phase in media with 0.02% MMS for an hour. Global gene expression analysis was performed using yeast microarrays. Genes down-regulated or up-regulated by INO80 over 4-fold are listed with the most affected genes on top and the least affected on bottom. Two tops genes, INO80 and TRP1 indicate the proper functioning of the microarray, since the ino80 stain lacks INO80, which was replaced by a TRP1 marker. None of the genes in the lists have known functions in DNA damage tolerance based on the functional classification of the Saccharomyces cerevisiae Database (SGD).

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Supplementary Table 2. Yeast strains used in this study.

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References 1 Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, et al. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 2003, 424: 1078-1083 2 Liberi G, Cotta-Ramusino C, Lopes M, Sogo J, Conti C, Bensimon A, Foiani M. Methods to study replication fork collapse in budding yeast. Methods Enzymol 2006, 409: 442-462 3 Tourriere H, Versini G, Cordon-Preciado V, Alabert C, Pasero P. Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol Cell 2005, 19: 699-706 4 Kao CF, Osley MA. In vivo assays to study histone ubiquitylation. Methods 2003, 31: 59-66 5 Hoege C, Pfander B, Moldovan GL, Pyrowolakis G, Jentsch S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 2002, 419: 135-141

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