Systematic analysis of dia2Δ synthetic genetic interactions. A total of 55 synthetic lethal (red edges) and synthetic sick genetic interactions (black edges) detected in triplicate SGA screens were confirmed by tetrad analysis. Interactions are grouped according to indicated cellular functions and individual nodes are colored by a reduced hierarchy of gene ontology (GO) biological processes ranked in the order shown in the color key.

Dia2 is required for resistance to agents that generate DNA adducts. The indicated strains were serially diluted in 10-fold steps and plated onto rich medium (A) or synthetic medium lacking tryptophan (B) in either the presence or the absence of 0.02% (v/v) MMS, 200 mm HU, 5 μg/ml camptothecin, 0.1 μg/ml 4-NQO, 200 J/m² of UV, or 100 Gy of X rays. Photographs were taken after 2 days at 30°.

Activation of the DNA damage response in the dia2Δ strain. (A) Cell cycle progression of unperturbed wild-type and dia2Δ strains. Cells were arrested in G₁ phase with α-factor and released into rich medium. DNA content was assessed by FACS at the indicated time points in minutes. (B) Rad53 kinase activity was detected using an ISA assay on lysates of the indicated strains grown in either the presence or the absence of 200 mm HU for 1 hr. Arrow indicates in situ ³²P incorporation into Rad53. (C) Cell cycle dependence of Rad53 activation. The indicated strains were arrested with α-factor and released into rich medium and lysates were prepared at the indicated time points. Immunoblots were probed with a polyclonal Rad53 antibody. (D) Cell cycle progression of MMS-treated wild-type and dia2Δ strains. Cells were arrested in G₁ phase with α-factor, released into rich medium containing 0.033% MMS, and assessed for DNA content at the indicated time points. (E) DNA damage repair foci in a dia2Δ strain. Rad52^YFP was detected by fluorescence microscopy in wild-type and dia2Δ cells. A composite of 21 collapsed Z-sections was taken for a minimum of 300 cells/strain, which were classified as unbudded (G₁) and budded (S/G₂/M) cells. Bars indicate standard error.

Increased GCR rate is correlated with hyperrecombination at the rDNA locus in dia2Δ cells. (A) Assay used to detect gross chromosomal rearrangements. Sensitivity to canavanine (can) and 5-FOA selects for colonies that have undergone simultaneous loss of both genes via a GCR event. GCR rates were from two independent experiments; fold induction was calculated as the mean of dia2Δ GCR rate divided by the mean GCR rate of the parental control strain. (B) Chromosomal DNA from a control and seven independent can^r 5-FOA^r dia2Δ isolates was resolved by PFGE, stained with ethidium bromide, and probed with a Chr V-specific MCM3 sequence. (C) Chromsomal DNA from five of the same isolates was rerun and probed with a 35S rDNA sequence. (D) Recombination at the rDNA locus. Wild-type and dia2Δ strains bearing an ADE2 marker at the rDNA locus were plated onto rich medium and grown for 3 days. Recombination rates were calculated from counting first division missegregation events for at least 20,000 colonies. Bars indicate standard error. (E) Accumulation of ERCs. Genomic DNA isolated from each of the indicated strains was resolved by electrophoresis and probed with rDNA sequences. Asterisk indicates chromosomal rDNA and arrows point to mono- and multimeric ERCs (red and black arrows, respectively). (F) Subcellular localization of Dia2^GFP. A wild-type strain expressing Dia2^GFP was grown to log phase and visualized by fluorescence microscopy. Dia2^GFP signal that segregates late in anaphase as a string of fluorescence stretched across the bud neck is indicated by the arrow. Numbers in parentheses indicate the percentage of cells that show nucleolar Dia2 localization at each cell cycle stage.

The F-box protein Dia2 forms an SCF complex and is required for normal cell cycle progression. (A) Physical interactions between Dia2^Flag and core components of the SCF complex. Cells were transformed with an empty vector or plasmids encoding Cdc4^FLAG or Dia2^FLAG expressed from the GAL1 promoter, in conjunction with a CEN plasmid that expressed Cdc53^MYC from the CDC53 promoter. Immunoblots of whole-cell lysates and anti-Flag immunoprecipitations were probed anti-Skp1 and anti-MYC antibodies. (B) Growth defect of dia2Δ strains. The dia2Δ spore clones from a sporulated heterozygous diploid DIA2/dia2Δ strain (yMT1738) are indicated by arrows. Cells from a representative tetrad were restreaked onto rich medium and grown at 30^o for 2 days. (C) Cell size distribution of dia2Δ strains. Spore clones from a DIA2/dia2Δ tetrad were grown to early log phase in liquid medium, analyzed on a Coulter channelizer, and visualized by DIC microscopy at ×100 magnification. (D) DNA content of asynchronous wild-type (WT) and dia2Δ populations. FACS profiles were deconstructed into G₁, S, and G₂/M components using ModFit LT software.

Two-dimensional hierarchical clustering of synthetic genetic interaction profiles. (A) A combined unique set of 144 genetic interactions from dia2Δ query screens reported in this study and in Pan et al. (2006) were clustered against 284 systematic genetic screens curated from the primary literature (Reguly et al. 2006). A locally dense region of interactions that contains the dia2Δ profile is expanded and immediate dia2Δ neighbors are indicated by the red bar. The source of each genetic interaction is indicated by the color key. (B) Shared interactions among dia2Δ, rtt101Δ, rtt107Δ,, and mms1Δ. Network shows all interactions retrieved from the full 284-screen data set for each of the four nodes. Edges are colored by interaction source; nodes are colored by the same GO biological processes as in Figure 2.

The DNA checkpoint is required for viability of a dia2Δ strain. (A) Representative tetrads were dissected for dia2Δ mec1Δ sml1Δ, dia2Δ rad53Δ chk1Δ sml1-1, dia2Δ rad9Δ rad24Δ, and dia2Δ mrc1Δ rad9Δ heterozygous diploids. Wild-type spores are unmarked, as are inviable spores whose genotype could not be inferred. The dia2Δ rad53Δ chk1Δ cross carries an unmarked sml1-1 allele to allow viability of the rad53Δ mutation. Note that, in the last tetrad shown, rad53Δ alone is inviable because sml1-1 did not segregate to this spore clone; however, specific inviability of dia2Δ rad53Δ sml1-1 was inferred from the viability of rad53Δ chk1Δ sml1-1 in the adjacent cross, as well as from other tetrads not shown. The mrc1Δ rad9Δ cross carries a <RNR1 LEU2 2μm> plasmid to maintain viability of the mrc1Δ rad9Δ double mutant. (B) Legend for determined genotypes. (C) Summary of genetic interactions. Minus sign indicates full inviability; plus sign indicates full viability. (D) Failure of various DNA damage checkpoint mutants to bypass the G₂/M cell cycle delay of dia2Δ strains. DNA content of asynchronous cultures of the indicated strains was determined by FACS analysis.

Dia2 is required to ensure faithful chromosome transmission. (A) Induction of RNR transcripts in a dia2Δ strain. Two replicate dia2Δ haploid isolates were competitively hybridized against wild-type control mRNA on genomewide microarrays (5989 ORFs detected) and plotted against one another. The top 10 induced genes in the dia2Δ strain (fold increase indicated in parentheses) were MET17 (6.5×), GPH1 (4.2×), HSP26 (3.4×), RNR4 (2.9×), YGP1(3.0×), RNR2 (3.4×), HSP12 (3.0×), YMR250w (3.1×), XBP1 (2.3×), and LAP4 (2.1×). Signals for RNR genes in replicate dia2Δ hybridizations are shown separately. (B) Aneuploidy in dia2Δ mutants detected by microarray analysis. Median gene induction per chromosome was calculated for independent haploid and homozygous diploid dia2Δ isolates. (C) Chromosome loss rates. Wild-type and dia2Δ strains bearing an artificial test chromosome (CFIII-SUP11-HIS3) were plated onto nonselective media for 2 days and scored for half red/white colonies (i.e., first division missegregation events). At least 2400 colonies were scored per strain.