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Fig. 7. Crt1 controls transcription of a suite of genes that may affect viability during replication stress. (A) Genome-wide transcriptional profiles were obtained by competitive hybridization of labeled cDNAs from ccr4 ${Delta}$ crt1 ${Delta}$ (MT3771, Cy5) vs. ccr4 ${Delta}$ (MT3768, Cy3) and for ccr4 ${Delta}$ dun1 ${Delta}$ crt1 ${Delta}$ (MT3773, Cy5) vs. ccr4 ${Delta}$ dun1 ${Delta}$ (MT3772, Cy3), as described in Materials and Methods. See Table 2 for specific gene induction/repression values. (B) Top differentially regulated genes between indicated strains, with and without HU treatment. Scale shows relative induction (red) or repression (green) with respect to control samples. (C) Effect of overexpression of RNR genes on sensitivity of the ccr4 ${Delta}$ dun1 ${Delta}$ double mutant to HU. (Top panels) Overexpression of RNR3. Strain ccr4 ${Delta}$ dun1 ${Delta}$ trp1 ${Delta}$ (MT3800) was transformed with either pGAP (pBAD054 TRP1, empty) or pGAP-RNR3 (pBAD079 TRP1, RNR3). ccr4 ${Delta}$ dun1 ${Delta}$ (MT3772), ccr4 ${Delta}$ dun1 ${Delta}$ crt1 ${Delta}$ (MT3773) and wild-type (BY4741) strains are shown for comparison. Strains were serially diluted, spotted on media lacking tryptophan (-trp) and containing indicated concentrations of HU and grown for 3-5 days. (Middle and bottom panels) Coordinate overexpression of multiple RNR subunits. Strain ccr4 ${Delta}$ dun1 ${Delta}$ trp1 ${Delta}$ (MT3800) was transformed with either pGAP (pBAD054 TRP1, empty vector) or pGAP-RNR3 (pBAD079 TRP1, RNR3) in combination with pGAL-RNR4 (pDL57 URA3, RNR4) and either pGAL-FLAG (pMT3164 LEU2, empty vector) or pGAL-RNR2-FLAG (pMT3963 LEU2, RNR2-FLAG). ccr4 ${Delta}$ dun1 ${Delta}$ crt1 ${Delta}$ (MT3773) was co-transformed with pGAL (pDL54 URA3, empty vector) or pGAL-FLAG (pMT3164 LEU2, empty vector) as a comparison. Strains were serially diluted, spotted on media lacking histidine, leucine, uracil and tryptophan (-HLUW) and containing indicated concentrations of HU and either glucose (GLU) or galactose (GAL) and grown for 3-5 days.

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