Synthetic dosage lethal screen by overexpression of SRS2, srs2-K41A and srs2-K41R in the strains of the yeast disruption library. (A) Centromeric overexpression plasmids containing SRS2, srs2-K41A or srs2-K41R under control of the CUP1 promoter. (B) Denaturing protein extracts from wild-type cells overexpressing Srs2 upon addition of 0, 50, 100, 200, 400 or 600 µM CuSO₄ to the growth medium. The extracts were migrated and revealed with an anti-Srs2 antibody and an anti-alpha-Tubulin antibody for a loading control. Asterisks mark post-translational modifications of the protein. (C) Manual verification of the SDL interactions with DNA metabolism genes. The interactions were found in the high-throughput screen and were verified by transforming the overexpression plasmids and an empty vector control into the deletion strains. The transformants were spotted in 10-fold serial dilutions onto medium without or with 100 µM CuSO₄. Overexpression in wild-type cells is presented as a control. (D) Heat-map representing the sensitivity of the DNA metabolism strains verified manually. For each strain, the three colour boxes correspond to the degree of its sensitivity to each of the three versions of SRS2: wild-type SRS2 or helicase-dead mutants srs2-K41A and srs2-K41R. Red: severe growth defect. Orange and Yellow: intermediate growth defects. The differences between these two classes are subtle and attributed according to growth of the each strain with the empty control vector. Green: same growth profile as with the empty control vector. * Genes that were not found in the high throughput screen.

Overexpression of wild-type or helicase-mutant SRS2 delays S-phase progression and acts independently of RAD51. (A) MATa bar1Δ cells containing an empty vector control or the plasmids for overexpression of SRS2 or srs2-K41A were synchronized in G1 with alpha factor. To induce overexpression, 200 µM CuSO₄ were added 30 minutes prior to release, and the induction was kept upon release. Samples were collected every 15 minutes for FACS analysis. (B) Cells were synchronized as described in (A) and samples were collected for protein extractions. The proteins were migrated on an acrylamide gel and revealed with antibodies againt Rad53 and alpha-Tubulin for a loading control. (C) Denaturing protein extracts of wild-type or rad51Δ cells overexpressing wild-type or helicase-dead SRS2. The extracts were revealed with antibodies against Rad53 and alpha-tubulin as a loading control. (B) rad51Δ, rad52Δ or rad51Δ rad52Δ mutants containing the overexpression plasmids were diluted in 10-fold serial dilutions and spotted onto medium without or with copper (100 µM and 200 µM CuSO₄).

Specificity and relevance of Srs2 overexpression. (A) srs2Δ cells transformed with an empty vector or with overexpression plasmids for SRS2, srs2-R1, srs2-K41A, srs2-K41A-R1, srs2-K41R and srs2-K41R-R1. 10-fold serial dilutions of the transformants were spotted on YPD as a spotting control and on medium without or with 100 µM CuSO₄. (B) Denaturing protein extracts of srs2Δ cells expressing SRS2, srs2-K41A, srs2-K41R, srs2-R1, srs2-K41A-R1 or srs2-K41R-R1 at 0 µM CuSO₄. Control extracts from srs2Δ cells with an empty vector and wild-type cells with an empty vector were used. The proteins were revealed with an anti-Srs2 antibody and anti-alpha-Tubulin antibody as a loading control. Total Srs2 protein was quantified relative to the amount of alpha-tubulin in each extract. (C) Overexpression of Elg1 does not induce a high Rad53 phosphorylation. Denaturing protein extracts of wild-type cells overexpressing SRS2 or ELG1-13MYC (three different clones) at 200 µM CuSO₄. *Control cells with endogenous tagged ELG1-13MYC. The proteins were revealed with ntibodies against Rad53, Srs2, Myc and alpha-Tubulin.

Overexpression of wild-type and helicase-dead SRS2 triggers the activation of the replication checkpoint. (A) mrc1Δ, csm3A, tof1Δ or rad9Δ mutants containing the overexpression plasmids were diluted in 10-fold serial dilutions and spotted onto medium without or with copper (100 µM CuSO₄). (B) Denaturing protein extracts of wild-type or mrc1Δ cells overexpressing wild-type or mutant SRS2 at 200 µM CuSO₄. The proteins were migrated on an acrylamide gel and revealed with antibodies against Rad53 and alpha-Tubulin for a loading control. (C) Rad53 phosphorylation. Native extracts of wild-type cells overexpressing wild-type or mutant SRS2. The extracts were either directly denatured after extraction (U: untreated), or incubated for at 1 hour at 30°C with phosphatase buffer, MnCl₂ and protease inhibitors (M: mock), or finally treated with λ-phosphatase for 1 hour at 30°C with phosphatase buffer, MnCl₂ and protease inhibitors (λ: λ-phosphatase treated). Note that the Rad53 is partially unphosphorylated in “Mock” samples suggesting the presence of phosphatases in the cellular extracts. (D) sml1Δ, mec1Δ sml1Δ, tel1Δ or mec1Δ tel1Δ sml1Δ mutants containing the overexpression plasmids were diluted in 10-fold serial dilutions and spotted onto medium without or with copper (100 µM CuSO₄).

The toxicity of Srs2 during replication depends on the domain that interacts with sumoylated PCNA. (A) mrc1Δ or rad27Δ cells transformed with an empty vector or with overexpression plasmids for SRS2, srs2-R1, srs2-K41A, srs2-K41A-R1, srs2-K41R and srs2-K41R-R1. 10-fold serial dilutions of the transformants were spotted on medium without or with copper (100 µM CuSO₄). (B) MATa bar1Δ cells containing an empty control vector or the plasmids for overexpression of srs2-K41A or srs2-K41A-R1 were synchronized in G1 with alpha factor. To induce overexpression, 200 µM CuSO₄ were added 30 minutes prior to release, and the induction was kept upon release. Samples were collected every 15 minutes for FACS analysis. (C) Protein extracts from wild-type cells overexpressing plasmids for SRS2, srs2-R1, srs2-K41A or srs2-K41A-R1, revealed with antibodies against Rad53 and alpha-Tubulin. 1 and 2 correspond to two independent transformants for srs2-R1 and srs2-K41A-R1. (D) Same as in (A) but with rad52Δ cells. (E) tda1Δ and fet3Δ cells transformed with an empty vector or with overexpression plasmids for SRS2 and srs2-R1. 10-fold serial dilutions of the transformants were spotted on medium without or with copper (200 µM CuSO₄).

Model representing the effects of overexpression of SRS2 and its helicase-dead mutants on DNA replication. ① Wild-type or helicase-dead SRS2 are recruited to replication forks by SUMOylated PCNA. This interaction likely prevents the recruitment of other factors required for replication, leading to fork progression delay, which activates the replication checkpoint (Mrc1, Tof1 and Csm3-mediated phosphorylation of Rad53). Cells overexpressing SRS2 eventually overcome this defect. The helicase-dead mutants srs2-K41A or srs2-K41R show a greater replication delay that likely leads to the accumulation of aberrant intermediates that require RAD52-dependent, RAD51-independent recombination to resume replication. ②The helicase-dead mutants induce activation of the DNA integrity checkpoint, resulting in hyperphosphorylation of Rad53. This effect is independent of DNA replication, is not associated with any damage and likely results from the accumulation of an Srs2 protein capable of triggering checkpoint activation.