a Drug concentration b 1.3% MMS nhp1 nhp1 8 nhp1 mag1.5% MMS.3% MMS nhp1 nhp1 ino8 9 ino8 9 % Viability 4.5% MMS ino8 9 ino8 9 2.5.1.15 % MMS c d nhp1 nhp1 nhp1 nhp1 nhp1 nhp1 Control (YPD) γ IR (1 gy) UV (2 J/m 2 ) 75mM HU.25µg/ml 4-NQO 5µg/ml CPT Drug concentration nhp1 nhp1 nhp1 nhp1 nhp1 nhp1 Supplementary Figure 1. Sensitivity of Isw2 and Ino8 complex mutants to inhibitors of replication. (a) MMS sensitivity of nhp1 and ino8 9 mutants. Wild type (W1588-4c), (YTT18), nhp1 (YTT2), nhp1 (YTT219), ino8 9 (YTT4179), and ino8 9 (YTT427) stationary phase cells were spotted across YPD plates containing gradients of MMS. The maximum concentration of the drug is indicated at the left. See Supplementary Methods for the protocol to make drug gradient plates. (b) Survival of nhp1 mutants during MMS treatment in culture. Strains as in (a) were grown to early log phase, treated with the indicated concentrations of MMS for one hour, then plated. Percent survival (+/- the standard deviation) of wild type, nhp1, and mag1 (YTT277, an MMS sensitive control strain) was determined by plating known numbers of cells before and after drug treatment and counting colonies. (c) Spot tests of UV and gamma irradiated cells. 1-fold serial dilutions of saturated cultures (a) were spotted to YPD plates then exposed to the doses of radiation indicated. Note that the number of colonies at each spot is comparable in each strain. (d) Sensitivity of nhp1 mutants to other DNA replication inhibitors. Stationary phase cells were spotted across YPD plates containing gradients of Hydroxyurea (HU), 4-nitroquinoline 1-oxide (4-NQO), or Camptothecin (CPT) as in (a).
Supplementary Figure 2. Replication profiles of MMS treated cells. (a) Schematic diagram of the dense isotope transfer experiments conducted in the presence of.15% MMS, and the generation of replication profiles from isotope labelled DNA collected throughout S phase. (b) FACS analyses of cell collection for generation of replication profiles. Wild type (YTT1831) and nhp1 (YTT336) cells were treated as outlined in (a) and collected for FACS at the indicated times after release from α-factor arrest. Samples used for generation of replication profiles shown in Figure 2 and in (c) are indicated by asterisks. Gray profiles are from cells collected prior to G1 arrest. (c) Replication profiles were generated from the samples indicated in (b). The collection of nhp1 mutants began at minutes post-release because of a slight delay in cell cycle initiation following alpha-factor arrest characteristic of the double mutant (b). Profiles from corresponding collection times are the same color. The total percentage of replication for the whole genome at each time-point was determined by slot blot of both replicated and unreplicated fractions used for microarray analysis, followed by hybridization with radioactively labeled and fragmented genomic DNA. Positions of confirmed and likely ARSs, defined at www.oridb.org, are indicated at the bottom of each graph. Filled triangles correspond to origins identified in two studies of replication timing 1,2 as being amongst the earliest 25% of origins replicated in a normal S phase. Open triangles represent origins that are amongst the earliest 25% in only one of the two studies. The filled circles correspond to remaining origins. Origins that are active in wild type cells treated with 2mM Hydroxyurea 3 are indicated in red. Positions of centromeres are indicated by the black circle on the x-axis. Replication profiles from all 16 chromosomes are displayed to facilitate comparison between genotypes
Supplementary Figure 2 a b 1) Grow in dense ( 13 C 15 N) media α-factor 15 min 2) Transfer to light ( 12 C 14 N) media α-factor 75 min MMS min Time 3 nhp1 3) Release in light media + MMS 4) Collect cells throughout S phase 5) Prepare and digest genomic DNA 6) Separate replicated DNA on CsCl gradient 7) Label replicated & unreplicated DNA replicated unreplicated 75 9 15 8) Compete replicated & unreplicated DNA on microarray 9) Generate replication profiles Chromosome Coordinate late-s mid-s early-s 12 135 15
c 1 8 Chr I 1 8 Supplementary Figure 2 Chr II 4 4 2 1 2 1 8 nhp1 4 2 1 2 2 1 2 3 4 5 7 8 1 8 nhp1 4 2 1 2 3 4 5 7 8 1 8 4 Chr III 1 8 4 Chr V time % total genomic replication nhp1 2 1 2 3 1 8 nhp1 2 1 2 3 4 5 1 8 nhp1 15 12 9 81% 66% % 35% 32% 27% 2% 4 4 31% 14% 2 1 2 3 2 1 2 3 4 5 3 12% 1 8 Chr IV 4 2 1 2 3 4 5 7 8 9 1 11 12 13 14 15 1 8 nhp1 4 2 1 2 3 4 5 7 8 9 1 11 12 13 14 15
c Supplementary Figure 2 1 8 Chr VI 1 8 Chr VII 4 4 2 1 2 1 8 nhp1 4 2 1 2 2 1 2 3 4 5 7 8 9 1 1 8 4 2 1 2 3 4 5 7 8 9 1 1 8 Chr VIII 1 8 Chr IX 4 4 2 1 2 3 4 5 1 8 nhp1 4 2 1 2 3 4 5 2 1 2 3 4 1 8 nhp1 4 2 1 2 3 4 1 8 4 Chr X time % total genomic replication nhp1 2 1 2 3 4 5 7 1 8 nhp1 15 12 9 81% 66% % 35% 32% 27% 2% 4 31% 14% 2 1 2 3 4 5 7 3 12%
c Supplementary Figure 2 1 8 4 Chr XI 2 1 2 3 4 5 1 8 nhp1 4 2 1 2 3 4 5 1 8 Chr XII 4 2 1 2 3 4 5 7 8 9 1 1 8 nhp1 4 2 1 2 3 4 5 7 8 9 1 1 8 4 Chr XIII time % total genomic replication nhp1 2 1 2 3 4 5 7 8 9 1 8 4 nhp1 15 12 9 81% 66% % 31% 35% 32% 27% 2% 14% 2 1 2 3 4 5 7 8 9 3 12%
c Supplementary Figure 2 1 8 4 Chr XIV 2 1 2 3 4 5 7 1 8 nhp1 4 2 1 2 3 4 5 7 1 8 Chr XV 4 2 1 2 3 4 5 7 8 9 1 1 8 nhp1 4 2 1 2 3 4 5 7 8 9 1 1 8 Chr XVI 4 2 1 2 3 4 5 7 8 9 1 8 nhp1 4 2 1 2 3 4 5 7 8 9 time 15 12 9 3 % total genomic replication 81% 66% % 31% 12% nhp1 35% 32% 27% 2% 14%
a Time post G1 release (+MMS): 3 9 12 Bubble arc (active origin) ARS7 (early) nhp1 Y arc d a 1n ARS1 (early-mid) nhp1 b S-phase (-MMS) ARS152 (late) nhp1 nhp1 ARS152 (late) ARS3 (late) nhp1 nhp1 ARS3 (late) Supplementary Figure 3. Patterns of origin firing in nhp1 mutants. (a) Origin firing during MMS treatment. The bubble arc is detected when an origin of replication is active in the probed restriction fragment. The ascending portion of the Y arcs ( a ; diagrammed on the right) arises when the probed region is replicated from forks originating outside of the restriction fragment, while the descending portion ( d ) can result from both passive replication and large replication bubbles from an active origin within the fragment. Genomic DNA for 2-D electrophoresis was isolated from (YTT1831) and nhp1 (YTT336) cells treated as in Supplementary Figure 2a. ARS7 is an early origin, ARS1 is an early-middle origin, while ARS152 and ARS3 initiate later in S phase. The grey arrows indicate faint bubble arcs at ARS3 and ARS152. Equivalent amounts of DNA were loaded as indicated by the intensity of 1N signals. See Supplementary Methods for the protocol. (b), 2-D gel analysis of late origin firing in the absence of MMS treatment. Genomic DNA for 2-D electrophoresis was isolated from strains undergoing S phase in the absence of MMS.
a 1 8 4 2 ARS7 DPB4 % budded 5 1 15 2 25 3 35 4 Time b Time 15 3 nhp1 15 3 1 8 4 2 nhp1 ARS7 DPB4 % budded 5 1 15 2 25 3 35 4 Time c 3 C 75 9 15 12 75 9 15 12 1 8 4 2 1 8 4 2 nhp1 135 15 135 15 d 2 4 8 1 12 2 4 8 1 12 Time Time 16 C 165 18 18 21 1 8 1 8 nhp1 195 24 4 2 4 2 21 3 5 1 15 2 25 3 Time 5 1 15 2 25 3 Time 24 3 Supplementary Figure 4. Fork progression analyses. (a) Comparison of replication kinetics of early and late-replicating loci during MMS treatment. Percent replication at an early-replicating locus, ARS7 (open circles), and a locus in a late-replicating region of Chromosome IV, DPB4 (Chromosome coordinate 694 kb, filled circles), was determined for each sample isolated in (b) as described in Methods. The percentage of budded cells is indicated by the dashed line. (b) FACS analysis of ARS8/ARS9 double deletion strains collected for fork progression-rate determination. Wild type (YTT3528) and nhp1 (YTT3531) cells were treated as outlined in Supplementary Figure 2a and collected for FACS analysis and isolation of genomic DNA at the indicated times after release from α-factor arrest. (c) Replication kinetics of the right arm of Chromosome VI at 3 C. Wild type and nhp1 cells, as in (b), were treated as in Supplemental Figure 2c except that MMS was omitted. Percent replication of regions 1-5 on chromosome VI, designated by blue lines of increasing intensity (as diagrammed in Figure 3a), was determined as described in Methods. The percentage of budded cells is indicated by the dashed line. The horizontal dotted line corresponds to the percent replication value designated as one-half of the maximum obtained for that experiment. The point of intersection of the horizontal dotted line with the fitted curve is designated as the T rep for that region. (d) Replication kinetics of the right arm of Chromosome VI at 16 C. As described in (c).
a 1) Grow cells to early log-phase 2) Split culture b vs. nhp1 nhp1 vs. nhp1 Untreated 2 hours 3 C.2% MMS Genes w/ 1.5 -fold change nhp1 specific changes Genes w/ 1.5 -fold change 3a) Harvest 3b) Harvest c log expression level nhp1 1 1 1 1 1 1 1 1 log expression level log expression level nhp1 1 1 1 1 1 1 1 1 log expression level nhp1 v nhp1 (MMS) (control) Supplementary Figure 5. Summary of gene-expression analysis of MMS-treated nhp1 mutants..1.1 1 1 1 1 1e4 s: JAV18 : MMS v MMS nhp1 (3 replicates) (Default Interpretation), v is... s: JAV18 : MMS v MMS nhp1 (3 replicates) (Default Interpretation), v is... Colored by: JAV18 : MMS v MMS nhp1 (3 replicates) (Default Interpretation) Gene List: all genes (68), 195 genes selected (a) MMS treatment and culture conditions. (YTT18), nhp1 (YTT2), and nhp1 (YTT219) strains were treated as shown before isolation of RNA. (b) Definition of nhp1 specific changes in gene-expression. Transcript levels in an nhp1 mutant were compared to transcript levels from either (blue) or nhp1 (orange) by co-hybridizing labeled mrna samples from the single and double mutant on the same microarray (indicated at the top of the diagram). Changes in transcript levels specific to the double mutant were defined as those common to both co-hybridization experiments. (c) Scatterplot of gene expression levels for MMS treated or nhp1 single mutants versus the expression levels of MMS treated nhp1 double mutants. The black data points correspond to 26 genes involved in replication, repair, or the DNA damage checkpoint. Blue lines mark 1.5-fold changes in gene expression. This gene list was compiled based on descriptions provided at the Saccharomyces Genome Database.