MMS-induced positive genetic interactions with RAD55 phosphorylation site mutants uncovers a novel link between Rad55 and nonsense-mediated decay (NMD). (A) Heat map displaying a subset of MMS-induced positive genetic interactions identified by E-MAP analysis and subsequently grouped by hierarchical clustering. (B) A network of previously curated physical protein interactions was generated using GeneMANIA for positively interacting array NMD genes identified in (A).

Validation of E-MAP results showing suppression of rad55 phosphorylation site mutants by nam7Δ using an independent strain background. (A) Plate assay measuring the sensitivity of W303 strains with the indicated genotypes to MMS. Serial dilutions (1:5) of cultures were spotted onto YPD containing 0.015% MMS and grown at 30°C for 2 days prior to imaging. (B) Plate assay as in (A). In order to assess the genetic interaction between rad55Δ and nam7Δ, a concentration of 0.008% MMS was used and plates were imaged after 3 days of growth.

Suppression of rad55-phosphorylation site mutants by nam7Δ is RAD51-dependent. Plate assays were performed with W303 strains containing the indicated mutations. (A) YPD plates containing 0% MMS and 0.005% MMS were incubated at 30°C for 2 days. (B) YPD Plates containing 0 and 0.01% MMS were imaged after 1 day of growth at 30°C.

Rad55 protein levels increase in NMD-deficient cells. (A) Representative immunoblots from mid-log cultures of WDHY2972 (RAD55-Myc9), WHDY3982 (RAD55-Myc9 nam7Δ), WDHY3449 (rad55-S2,8,14,19,20A-Myc9) and WDHY3980 (rad55-S2,8,14,19,20A nam7Δ). Cultures were either left untreated or treated with 0.075% MMS for 1 h. Antibodies against the Myc epitope were used to determine Rad55 levels. Membranes were reprobed with antibodies against Pgk1, which served as a loading control. (B) Plots of quantified immunoblot signals by densitometry. Rad55 signals were normalized to Pgk1 level from the same sample. The average normalized Rad55 levels of at least three experiments were plotted with error bars representing standard error of the mean. The Rad55 (upper panel) and Rad55-S2,8,14,19,20A (lower panel) samples were quantified from different exposures (see Supplementary Figure S6) and plotted in separate graphs.

Rad55 mRNA levels are regulated by NMD. (A) qPCR using primers specific to the intron region of CYH2 were used as a readout for NMD activity in NAM7 (WDHY2217) and nam7Δ (WDHY3865) strains. CYH2 levels were calculated relative to ScR1 transcript levels which are unaffected by NMD and serve as a baseline control for normalization. Each data point is the average of three independent experiments. Error bars represent standard error of the mean. Statistical significance was calculated by a two-tailed t-test and defined by a P-value < 0.05. (B) RAD55 mRNA levels were measured by qPCR using the same strains as in (A). Cells were grown to mid-log and treated with 0.075% MMS for one hour where indicated. Error bars represent standard error of the mean. Statistical significance was calculated by a two-tailed t-test and defined by a P-value < 0.05. (C) rpb1–1 shutoff experiment measuring RAD55 mRNA decay. Plotted are the average levels of RAD55 mRNA relative to ScRI as measured by qPCR in NAM7 (WDHY4057) and nam7Δ (WDHY4103) strains. Each data point represents the average of three independent samples collected at the indicated time after shifting cultures to the non-permissive temperature of 39°C. Non-linear regression using a first-order decay equation (solid lines, dotted lines represent the 95% confidence interval) was used to calculate the half-life of RAD55 mRNA in wild-type and nam7Δ strains.

NMD regulates the mRNA levels of multiple genes involved in homologous recombination (HR). qPCR was used to measure mRNA levels of RAD51, RAD57, RAD52 and RAD54 in WDHY2217 (NAM7) and WDHY3865 (nam7Δ). Relative mRNA levels were calculated by normalizing to ScRI RNA levels. Each plot represents the average of at least three individual experiments, error bars represent standard error of the mean. Statistical significance was calculated by a two-tailed t-test and defined by a P-value < 0.05.

In vivo consequences of NMD on DNA damage resistance and HR. (A) Plate assay measuring the sensitivity of wild-type (WDHY2217), nam7Δ (WDHY3865) and nmd2Δ (WDHY4012) strains to MMS. Serial dilutions (1:5) of cultures were spotted onto YPD containing 0.04% MMS and grown at 30°C for 4 days prior to imaging. (B) Plot showing Ade⁺ Ura⁺ recombination rates of the indicated strains (wild-type: WDHY2371; nmd2Δ: WDHY4650; nam7Δ:WDHY4660). Rates were defined as the number of events/cell/division. Error bars represent the 95% confidence interval, non-overlapping of error bars between two samples was deemed statistically significant.