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. 2015 Apr 6;290(20):12664–12675. doi: 10.1074/jbc.M115.645408

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — Characterization of the ddi2/3 deletion mutants. A, schematic diagrams of DDI2/3 gene deletion constructs. B, yeast genomic PCR confirms the disruption of DDI2 and/or DDI3 genes. Lane M, 1-kb DNA ladders (from Invitrogen); lane 1, BY4741; lane 2, WXY3148 (ddi2/3Δ::LEU2); lane 3, WXY3147 (ddi2/3Δ::HIS3); lane 4, WXY2929 (ddi2/3Δ::LEU2 ddi2/3Δ::HIS3). Primers are DDI2–4 and DDI2–5, flanking the DDI2/DDI3 ORFs. C, serial dilution assay of ddi2/3Δ cells to test sensitivity to cyanamide (Cy) or MMS. Overnight yeast cultures were normalized, and diluted cells were equally spotted on YPD and cyanamide- or MMS-containing YPD plates. D, serial dilution assay of the mutants with disrupted duplicated chromosomal regions and/or ddi2/3 mutations showing killing by cyanamide or MMS. E, serial dilution assay of ddi2/3Δ cells showing sensitivity to cyanamide at higher concentrations. Plates shown in C–E were incubated at 30 °C for 3 days before being photographed.