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. 2017 Mar 17;45(5):2558-2570.
doi: 10.1093/nar/gkw1249.

Degradation of Mrc1 promotes recombination-mediated restart of stalled replication forks

Indrajit Chaudhury  1 , Deanna M Koepp  1
Affiliations

Degradation of Mrc1 promotes recombination-mediated restart of stalled replication forks

Indrajit Chaudhury et al. Nucleic Acids Res. .

Abstract

The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. Replication stress or DNA damage triggers fork stalling and checkpoint signaling to activate repair pathways. Recovery from checkpoint activation is critical for cell survival following DNA damage. Recovery from the S-phase checkpoint includes inactivation of checkpoint signaling and restart of stalled replication forks. Previous studies demonstrated that degradation of Mrc1, the Saccharomyces cerevisiae ortholog of human Claspin, is facilitated by the SCFDia2 ubiquitin ligase and is important for cell cycle re-entry after DNA damage-induced S-phase checkpoint activation. Here, we show that degradation of Mrc1 facilitated by the SCFDia2 complex is critical to restart stalled replication forks during checkpoint recovery. Using DNA fiber analysis, we showed that Dia2 functions with the Sgs1 and Mph1 helicases (orthologs of human BLM and FANCM, respectively) in the recombination-mediated fork restart pathway. In addition, Dia2 physically interacts with Sgs1 upon checkpoint activation. Importantly, failure to target Mrc1 for degradation during recovery inhibits Sgs1 chromatin association, but this can be alleviated by induced proteolysis of Mrc1 after checkpoint activation. Together, these studies provide new mechanistic insights into how cells recover from activation of the S-phase checkpoint.

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Figures

Figure 1.
Figure 1.
Dia2 is essential for restart of stalled replication forks. (A) Ten fold serial dilutions of the indicated strains were spotted on yeast extract-peptone-dextrose (YPD), YPD + 0.007% methyl methane sulfonate (MMS) or 200 mM hydroxyurea (HU) and incubated at 30°C. (B) Equal number of cells were plated on media containing the indicated amounts of MMS or HU and colony-forming units were counted after 4 days at 30°C. Error bars represent standard deviations from three independent experiments. (C) Schematic of DNA fiber assay depicting sites of replication. Red tracts, IdU; green tracts, CldU. (D) Representative figure showing inability to restart replication after 0.05% MMS treatment in absence of Dia2, Sgs1 or Mph1. (E) Dia2, Sgs1 and Mph1 work in concert to mediate replication fork restart after MMS-induced fork stalling. The replication restart efficiencies of wild type, dia2Δ, sgs1Δ, mph1Δ, dia2Δsgs1Δ and dia2Δmph1Δ strains were measured as the number of restarted replication forks (IdU–CldU tracts) compared with the total number of IdU-labeled tracts (IdU only tracts plus IdU–CldU tracts). ***P < 0.001.
Figure 2.
Figure 2.
Dia2 and Sgs1 maintains normal replication velocity and F-Box domain of Dia2 is important for both fork restart as well as fork speed. (A) Distributions of CldU tract lengths were determined on IdU–CldU double-labeled DNA fibers isolated from untreated wild type, dia2Δ, sgs1Δ, mph1Δ, dia2Δsgs1Δ and dia2Δmph1Δ strains. P < 0.05 for wild type versus dia2Δ, sgs1Δ, dia2Δsgs1Δ or dia2Δmph1Δ strains and P-value is not significant for wild type versus mph1Δ strain. (B) Replication fork restart efiiciencies of wild type, dia2Δ and the dia2 F-Box domain deletion strains (dia2ΔF) were measured as the number of restarted replication forks (IdU–CldU tracts) compared with the total number of IdU-labeled tracts (IdU only and IdU–CldU tracts). ***P < 0.001. (C) CldU tract length distributions were determined in wild type, dia2Δ and dia2 F-Box domain deletion strains (dia2ΔF). P < 0.05 for wild type versus dia2Δ or dia2ΔF.
Figure 3.
Figure 3.
Mph1 protects nascent DNA strands from nucleolytic degradation after fork stalling. Nascent replication fork tract lengths (labeled with IdU only) were measured after 1 h of 0.05% MMS treatment in dia2Δ, sgs1Δ, mph1Δ, dia2Δsgs1Δ and dia2Δmph1Δ strains. P < 0.05 for wild type versus dia2Δ, sgs1Δ or dia2Δsgs1Δ and P < 0.001 for wild type versus mph1Δ or dia2Δmph1Δ strains.
Figure 4.
Figure 4.
Dia2-Sgs1 interaction is induced by stalled replication forks. (A) Dia2 physically interacts with Sgs1. Untreated (NT), 0.05% MMS treated or 200 mM HU treated cell lysates prepared from wild type as well as 9myc-tagged Dia2 strains were used in anti-myc immunoprecipitations (lanes 8, 10, 12, 14, 16 and 18) or incubated with protein A/G beads without antibody as a negative control (lanes 7, 9, 11, 13, 15 and 17). Immunoblots were probed with anti-myc or anti-Sgs1 antibodies. (B) Dia2 interaction with Sgs1 is not via DNA bridges. Cell lysates from wild-type cells (lane 1) were either untreated or treated with DNaseI prior to immunoprecipitation with anti-Myc antibody. (C) Sgs1 interacts with the TPR domain in Dia2. (i) Schematics showing Dia2 protein domains and mutants used in the immunoprecipitation study. TPR, tetratricopeptide repeats; F, F-Box domain; LRR, leucine-rich repeats. (ii) Strains expressing Myc-tagged Dia2 protein segments were treated with 0.05% MMS. Cell lysates (lanes 11–15) were prepared for immunoprecipitations with an anti-myc antibody (lanes 1–5) or protein A/G beads without antibody (lanes 6–10). Immunoblots were probed with anti-myc and anti-Sgs1 antibodies.
Figure 5.
Figure 5.
An N-terminal domain of Dia2 is required for checkpoint recovery. (A) Schematic showing N-terminal deletion mutants of Dia2. (B) Cells were arrested in G1 phase of cell cycle by α-factor (αF), released into YPD + 0.033% MMS for 40 min followed by release into YPD without MMS for indicated time points to allow checkpoint recovery. Samples were analyzed by flow cytometry. 1C and 2C indicate DNA content and percentage of cells with 2C DNA content is shown on the right side of the selected profiles.
Figure 6.
Figure 6.
Upon stalled fork recovery, Dia2 dissociates from Sgs1. Wild-type cells were either untreated or treated with 0.05% MMS for 20 or 40 min and allowed to recover in the fresh media for 20, 40 and 60 min. Whole cell lysates were prepared and used in immunoprecipitations with an anti-myc antibody (lanes 1–9) or protein A/G beads without antibody (lanes 10–18). Immunoblots were probed with anti-myc or anti-Sgs1 antibodies.
Figure 7.
Figure 7.
Depletion of Mrc1 in dia2Δ strains rescues deficiencies in fork restart and maintenance of replication velocity by recruiting Sgs1 to chromatin. (A) Replication fork restart efficiencies of wild-type and a dia2Δmrc1 degron strain with or without IAA were measured as number of restarted forks (IdU-CldU tracts) compared with the total number of IdU-labeled tracts (IdU only plus IdU–CldU tracts). ***P < 0.001, NS = not significant. (B) CldU tract length distributions were determined in wild type, and a dia2Δmrc1 degron strain with or without IAA. P < 0.05 for wild type versus dia2Δmrc1 degron - IAA, and P-value is not significant for wild type versus dia2Δmrc1 degron + IAA. (C (i) and (ii)) Crude pellet containing chromatin fractions were separated from soluble cytoplasmic fractions from the untreated (lanes 1–6) or DNaseI-treated (lanes 7–12) whole cell lysates of wild type and a dia2Δmrc1 degron strain with or without IAA. Samples were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for Dia2Myc, Mrc1HA and Sgs1 using anti-Myc, anti-HA or anti-Sgs1 antibodies, respectively. S, soluble cytoplasmic fraction; P, pellet containing chromatin fraction.
Figure 8.
Figure 8.
Model depicting role of Dia2 in replication fork restart. Dia2 mediates stalled replication fork recovery by promoting recruitment of Sgs1 and degradation of Mrc1. In response to MMS induced replication fork stalling, Dia2 interacts with Sgs1. Dia2 and Sgs1 work in concert to restart stalled fork. Dia2 mediates ubiquitination of Mrc1 to facilitate Mrc1 degradation via the proteasome. In the absence of Dia2, Mrc1 degradation, Sgs1 chromatin association and replication fork recovery are inhibited. These defects can be reversed by induced degradation of Mrc1 in dia2Δ cells.
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