Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Nov;201(3):937-49.
doi: 10.1534/genetics.115.181479. Epub 2015 Sep 11.

Termination of Replication Stress Signaling via Concerted Action of the Slx4 Scaffold and the PP4 Phosphatase

Affiliations
Free PMC article

Termination of Replication Stress Signaling via Concerted Action of the Slx4 Scaffold and the PP4 Phosphatase

Carolyn M Jablonowski et al. Genetics. .
Free PMC article

Abstract

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.

Keywords: DNA damage checkpoint; PP4; Rad53; Slx4; replication stress.

Figures

Figure 1
Figure 1
Cells lacking either PPH3 or SLX4 show similar defects upon replication stress induced by MMS. (A) Serial dilution assays showing the effect of genotoxin treatment upon the sensitivity of wild-type, slx4Δ, and pph3Δ cells. Fourfold serial dilutions were spotted on YPD plates and grown for 2–3 days at 30°. (B) Anti-Rad53 immunoblots of wild-type, slx4Δ, and pph3Δ strains showing Rad53 phosphorylation status after MMS treatment. (C) S-phase progression analysis of wild-type, slx4Δ, and pph3Δ strains. For B and C, cells were arrested in G1 with α-factor and then released in medium containing MMS. Samples were collected in G1 and at different time points following release. (D) Analysis of fully replicated chromosomes measured by PFGE in wild-type, slx4Δ, and pph3Δ strains. Asynchronous (ASY) cells were treated with 0.01% MMS for 2 hr and then released in MMS-free medium for up to 6 hr. (E) Serial dilution assay showing the effect of a hypomorphic RAD53 allele (rad53-R605A) on MMS sensitivity of wild-type, slx4Δ, and pph3Δ strains.
Figure 2
Figure 2
Slx4 and Pph3 function in a complementary manner in the regulation of Rad53 signaling. (A–C) Wild-type, slx4Δ, and pph3Δ single mutants were compared against a pph3Δ slx4Δ strain. (A) Anti-Rad53 immunoblots of wild-type, slx4Δ, pph3Δ, and pph3Δ slx4Δ strains showing Rad53 phosphorylation status after MMS treatment. (B) Serial dilutions assay showing the MMS sensitivity of indicated strains. (C) Analysis of fully replicated chromosomes by PFGE. Asynchronous (ASY) cells were treated with 0.005% MMS for 2 hr and then released in MMS-free medium for up to 5 hr. (D–G) Effect of the rad53-R605A allele on (D) Rad53 phosphorylation status, (E) MMS sensitivity, (F) PFGE-monitored chromosomes, and (G) S-phase progression of pph3Δ slx4Δ cells. For B and E, assays were performed as described in Figure 1A.
Figure 3
Figure 3
Rad53 hypersignaling in cells lacking PPH3 and/or SLX4 converges to misregulation of the Mus81-Mms4 endonuclease. (A and B) Serial dilution assay showing the effect of MUS81 or SGS1 deletion on the MMS sensitivity of the indicated strains. (C–E) Effect of the rad53-R605A allele on (C) MMS sensitivity, (D) S-phase progression, and (E) PFGE-monitored chromosomes of the indicated strains lacking MUS81. In D, cells were released from G1 arrest in YPD medium containing 0.015% MMS. In E, asynchronous cells were treated with 0.01% MMS for 2 hr and released into MMS-free medium for up to 5 hr. Samples were taken at each indicated time point. (F) Serial dilution assay showing the effect of SGS1 deletion on the MMS sensitivity of a pph3Δ slx4-S486A strain.
Figure 4
Figure 4
Antagonistic roles for γ-H2A in DDC control. (A and B) Effect of an H2A phospho-mutant (hta-S129A) on the MMS sensitivity (A) and Rad53 phosphorylation (B) of slx4Δ and pph3Δ strains. In B, asynchronous cells were treated with 0.005% MMS for 2 hr and samples were collected. Western blot analyses were performed using anti-Rad53 and anti-γ-H2A antibodies as described in Material and Methods. (C) Serial dilution assay showing the effect of DOT1 deletion on the MMS sensitivity of slx4Δ and pph3Δ strains. (D) Proposed model conciliating the antagonistic roles of γ-H2A on DDC regulation.
Figure 5
Figure 5
Pph3 and Slx4 function in spatially distinct manners. (A) Representative images showing the intracellular localization of Slx4 and Pph3 proteins. (B) Slx4-yEmCherry and Php3-GFP foci were measured by confocal fluorescence microscopy after MMS treatment. The percentage of cells with Slx4-yEmCherry, Php3-GFP, and both Slx4-yEmCherry/Php3-GFP foci is plotted. (C) Serial dilution assay showing the effect of MBD expression on the MMS sensitivity of the selected strains. MBD and SLX4 were expressed from a pRS416 plasmid (for details see Table S2) in SC –URA. (D–F) ChIP-seq analysis was performed following synchronous release of wild-type (D), pph3Δ (E), and slx4Δ (F) cells into S phase in the presence of 0.04% MMS for 60 min. γ-H2A (S129) enrichment scores on chromosome X are shown. Early origins are indicated by green bars and late origins by red bars. (G and H) The median γ-H2A ChIP enrichment scores (G) and replication profiles (H) across 108 early-firing origins, in wild-type, pph3Δ, and slx4Δ cells, are plotted. (I) Immunoblot showing the status of Rad53 and γ-H2A in wild-type, pph3Δ, and slx4Δ cells after treatment of G1 synchronized cultures with α-factor and release into medium containing 0.01% MMS for the indicated time points. (J) Proposed model illustrating how Pph3 and Slx4 coordinate Rad53 downregulation in spatially distinct manners.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback