(A) Representative growth curves of 16 individual strains grown in the presence of solvent alone (DMSO, red curve) and 62.5 μM mechlorethamine (black curve). Growth was monitored by measuring the optical density (OD₆₀₀) of cultures every 15 min for 30 h. The fitness of each strain was defined by the difference between the average doubling times in mechlorethamine and in DMSO (see Materials and Methods).
(B) Correlation between growth rates of individual strains and microarray-based fitness estimates. The ratio of growth rates of the 186 individual homozygous deletion strains (the top 233 ranked mechlorethamine-sensitive strains as determined by three replicate microarray experiments minus 47 strains which exhibited a slow-growth phenotype when individually cultured in the absence of mechlorethamine) over an average wild-type growth rate are plotted on the x-axis against the average fitness-defect scores from three pool experiments on the y-axis. The correlation (R² = 0.7462) is highly significant (p = 5.4e−57).

Detailed examination of strains with mutations in DNA-damage-response genes. Each bar graph represents only strains that were found to be among the top 30 (or 250) most sensitive strains in that compound and are known to be members of a well-characterized DNA-damage-response pathway. The bars represent the median rank for genes in each of the gene groups listed in the visual key. The gene groups were defined in the following way: x-linking genes (PSO2); NER (RAD2, RAD4, RAD10, RAD1, and RAD14); PRR (RAD6, RAD18, and RAD5); error-prone TLS (REV1, REV3, and REV7); HRR (RAD57, RAD55, RAD51, RAD52, RAD54, and RAD59); stalled replication-fork repair (MUS81 and MMS4). Those compounds that form ICLs are labeled with an asterisk.

(A) Pie charts showing relative percentage of sensitive genes categorized into either “DNA metabolism”, “unknown”, or “other”. All of the Gene Ontology [63] slim annotations (ftp://ftp.yeastgenome.org/yeast/data_download/literature_curation/go_slim_mapping.tab. Accessed February 17, 2005) are combined into “other” except for those classified in the unknown or DNA-metabolism category. The relative distributions of the mechlorethamine experiments are shown as a function of both time and gene rank.
(B) BY4743 (wild-type) and the DNA-damage-checkpoint mutants, rad9Δ and rad24Δ, were grown in the presence of 500 μM cisplatin over the course of ten population doublings. Yeast cultures were maintained in an exponential phase of growth by robotic dilution of cultures after five doublings into fresh media containing cisplatin. The growth of each deletion strain (black curve) is compared to that of BY4743 (red curve) between 0–5 (left) and 6–10 (right) population doublings. The rad9Δ and rad24Δ deletion strains exhibit accelerated grow rates in the first five generations, but by ten generations begin to demonstrate slow growth as compared to wild-type.
(C) Viability assay of strains treated with cisplatin or DMSO for five generations. Indicated strains were removed from cultures after they reached an OD₆₀₀ of 2.0, and were diluted as shown. Strains were chosen based on the criteria that they did not show a growth defect at five generations but did show a growth defect at 20 generations. Dilutions were pinned onto YPD plates in a 5-fold concentration series. The wild-type parental diploid strain is compared to several diploid deletion strains that exhibited a decrease in viability: ddc1Δ, rad24Δ, rad17Δ, mec3Δ, and rad9Δ. In contrast, several of these strains showed little or no decrease in viability at five generations of growth: swi4Δ, yme1Δ, rtf1Δ, and gcs1Δ. This figure underscores the point that, despite the lack of an apparent growth defect in liquid culture, several deletion strains lose viability relatively rapidly when exposed to cisplatin.

(A) Clustergram containing all strains significant in two or more array experiments. Raw fitness-defect values were hierarchically clustered using Spearman's rank correlation. Colored bars represent gene clusters of note, including NER (RAD2, RAD4, RAD10, RAD14, and RAD1—blue); error-prone TLS (REV1 and REV3—red); PRR (RAD6, RAD18, and RAD5—yellow); homologous recombination (RAD57, RAD51, and RAD54—green); cell-cycle checkpoint control (RAD9, RAD24, RAD17, DDC1, and MEC3—orange); and a cluster shown in (B) (SHU2, SHU1, CSM2, MPH1, and PSY3—magenta).
(B) Zoom view showing one cluster containing the class I NER genes and a second cluster containing several uncharacterized DNA-repair genes. Four of these five genes (SHU1, SHU2, CSM2, and PSY3) are known to encode proteins that physically interact [65,77,78].
(C) Individual growth curves of single and double deletion strains with MPH1 in 0.002% MMS. In all panels, the growth of wild-type (BY4741) is represented by the black curve and the growth of mph1Δ by the red curve. The growth of eight different deletion strains (shu1Δ, shu2Δ, csm2Δ, psy3Δ, mag1Δ, mus81Δ, rad51Δ, and rad54Δ) are shown in green, and double mutants, in which the MPH1 deletion is added to each of the above, are shown in blue. Double mutants of MPH1, MAG1, and MUS81 show additive or synergistic sensitivity to MMS, whereas double mutants of MPH1, with the four other genes in its cluster, show no additional sensitivity to MMS, suggesting that MPH1 is epistatic with SHU1, SHU2, CSM2, and PSY3.