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. 2019 Nov 25;15(11):e1008427.
doi: 10.1371/journal.pgen.1008427. eCollection 2019 Nov.

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Alicja Winczura  1 , Rowin Appanah  1 , Michael H Tatham  2 , Ronald T Hay  2 , Giacomo De Piccoli  1
Affiliations

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Alicja Winczura et al. PLoS Genet. .

Abstract

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pol2 is mono-SUMOylated on chromatin in response to nucleotide depletion.
A) Pol2 is post-translationally modified in response to replication stress, especially following treatment with HU. Cells were grown to the exponential phase, arrested in G1 and synchronously released in S phase for 30 min in YPD (S phase), or for 90 min in medium containing 0.2 M HU (HU), 0.033% methyl methanesulphonate (MMS) or 20 μM Camptothecin (CPT). Exponentially growing cells were also arrested at the G2/M phase with nocodazole, and incubated in the absence (Nz) or in the presence of 70 μM Zeocin (Nz+NEO) for 90 min. Rad53 and Pol2 immunoblotting are shown. B) Pol2 is SUMOylated in response to HU. Cells carrying a TAP-tagged version of Dpb2 were grown to exponential phase, arrested in G1 and synchronously released in S phase for 30 min in YPD (S phase) or for 90 min in YPD 0.2 M HU (HU). Pol ε was purified under stringent conditions (700 mM potassium acetate) and the immunoprecipitated material was eluted by TEV cleavage of the TAP tag. Cell extracts and IPs were probed with an anti-SUMO antibody. C) SUMOylated Pol2 is enriched at forks. Cells carrying a FLAG-tagged allele of Cdc45 were grown to exponential phase, arrested in G1 and synchronously released in YPD 0.2 M HU (HU) for 90 min. Proteins were cross-linked with formaldehyde. Cdc45 was immunoprecipitated and protein samples were analysed by immunoblotting. D) Pol2 is mono-SUMOylated. (Top). Schematic representation of the tagged alleles of POL2 used. (Bottom). Cells carrying a TAP-tagged version of Dpb2 and either a wild type, a FLAG-tagged or a SUMO-tagged versions of POL2 were grown to exponential phase, arrested in G1 and synchronously released in YPD 0.2 M HU for 90 min. Dpb2 was then purified and analysed by immunoblotting.
Fig 2
Fig 2. Pol2 is the major target of SUMOylation within the replisome in response to nucleotides depletion.
A) Pol2 is the major SUMOylation substrate among the replicative DNA polymerases in response to HU. Cells carrying a TAP-tagged version of Dpb2 (Pol ε), Pol12 (Pol α) or Pol31 (Pol δ) were grown to the exponential phase, arrested in G1, and synchronously released in S phase in the absence (S) or in the presence of 0.2 M HU for 30 min and 90 min, respectively. The TAP-tagged proteins were immunoprecipitated under stringent conditions, eluted by TEV cleavage of the TAP tag and analysed by immunoblotting. The samples were also analysed by mass spectrometry and shown to co-purify all components of the three polymerases (S2 Fig). B) Pol2 is the main target of SUMOylation within the replisome in response to HU. Cells carrying a TAP-tagged version of Dpb2 (Pol ε) or Sld5 (GINS) were synchronously released in the absence (S) or in the presence of 0.2 M HU for 30 min and 90 min, respectively. The TAP-tagged proteins were immunoprecipitated from 2.5*109 (Dpb2-TAP) and 1010 cells (TAP-Sld5) in 300mM potassium acetate, eluted by TEV cleavage of the TAP tag and (for the Sld5 IPs) concentrated by TCA precipitation. TAP-Sld5 purification allows to isolate the Replisome Progression Complex, comprising of the CMG helicase and other regulatory factors at the replication fork (114). Protein samples were separated by electrophoresis and Coomassie-stained or analysed by immunoblotting. The symbol < indicates the TEV proteinase band, the symbol * indicates the un-cleaved form of TAP-Sld5.
Fig 3
Fig 3. Pol2 SUMOylation depends on Rad53 and S phase checkpoint mediators.
A) Representation of the checkpoint kinases cascade. B) Pol2 SUMOylation depends on the checkpoint kinases Mec1 and Rad53. Strains sml1Δ (WT), sml1Δ mec1Δ (mec1Δ), sml1Δ rad53Δ (rad53Δ) and sml1Δ dun1Δ (dun1Δ), all carrying a TAP-tagged version of DPB2, were grown to the exponential phase, arrested in G1 and synchronously released in medium containing 0.2 M HU for 90 min. Dpb2 was immunoprecipitated and the protein samples were analysed by immunoblotting. C) Pol2 SUMOylation depends on the S phase checkpoint mediators Mrc1 and Ctf18. Strains deleted for genes required for the activation of Rad53 in response to double strand breaks (mre11Δ, rad50Δ), to replication stress, either through the S phase checkpoint (mrc1Δ, ctf18Δ, mrc1-AQ), or the DNA damage checkpoint (rad9Δ), were grown to the exponential phase, arrested in G1 and synchronously released in medium containing 0.2 M HU for 90 min. Pol ε was immunoprecipitated via Dpb2-TAP and analysed by immunoblotting. D) Analysis of the kinetics of Pol2 SUMOylation. Wild type cells, mutants sld3-37A dbf4-4A, and sml1Δ rad53Δ were grown to the exponential phase, arrested in G1 and synchronously released in medium containing 0.2 M HU for 90 min. Cells were collected every 30 min, and proteins were analysed by TCA extraction and immunoblotting. Pol2 SUMOylation appears after Rad53 activation and it’s more pronounced in cells defective in the inhibition of late origin firing.
Fig 4
Fig 4. Pol2 is SUMOylated by Mms21 and interacts with the Smc5/6 complex.
A) Pol2 SUMOylation depends on the E3-SUMO ligase Mms21. Wild type, siz1Δ and siz2Δ cells were grown to the exponential phase, arrested in G1 and synchronously released in medium containing 0.2 M HU for 90 min. In addition, a control strain (Ctr) and one carrying an auxin-inducible degron allele of MMS21 (mms21), were grown in YPRaf to the exponential phase, arrested in G1, resuspended in YPGal for 35 min, incubated for 60 min in medium containing a final concentration of 0.5 mM indole-3-acetic acid (IAA) to induce protein degradation, and released in YPGal containing 0.2 M HU and 0.5 mM IAA for 90 min. Dpb2-TAP was immunoprecipitated and protein samples were analysed by immunoblotting. B) Smc5/6 complex is required for Pol2 SUMOylation. Strains wild type, mms21-aid (mms21), smc5-aid (smc5), scc2-aid scc4-aid (scc2 scc4) and a control strain were grown in YPRaf to the exponential phase, arrested in G1, resuspended in YPGal for 35 min, incubated for 60 min in medium containing a final concentration of 0.5 mM IAA, and released in YPGal medium containing 0.2 M HU and 0.5 mM IAA for 90 min. Dpb2-TAP was immunoprecipitated and protein samples were analysed by immunoblotting. C) Pol2 is SUMOylated in vitro by Mms21. Pol ε and the Smc5/6 complex—either carrying a wild type allele of MMS21 or the SUMO-ligase defective mms21-(C200A H202A) mutant, (referred to as mms21-CH, also see S4B Fig)—were purified at high salt conditions (700 mM potassium acetate) via a TAP tag (on Dpb2 and Smc5 respectively). SUMO (Smt3GGΔ), the E1 SUMO-activating enzymes Aos1/Uba2 and the E2 SUMO-conjugating enzyme Ubc9 were purified from E. coli. The in vitro SUMOylation reaction was conducted for 60 min at 30°C. D) Pol2 SUMOylation partially depends on RTT107 and ESC2. An untagged strain (Ø), or wild type, rtt107Δ and esc2Δ cells carrying a TAP-tagged allele of DPB2, were grown to the exponential phase, arrested in G1 and synchronously released in medium containing 0.2 M HU for 90 min. Dpb2-TAP was immunoprecipitated and protein samples were analysed by immunoblotting. E) Smc5 co-immunoprecipitates with Pol ε in G1 and S phase. Cells carrying either a tagged or untagged version of Dpb2 were grown to exponential phase, arrested in G1 and released in YPD for 30 min (S) or in YPD 0.2 M HU for 90 min (HU). Dpb2-TAP was immunoprecipitated and protein samples were analysed by immunoblotting. F) Pol2 co-immunoprecipitates with Mms21. Cells carrying a tagged or untagged version of Mms21 were arrested in G1 and released in YPD 0.2 M HU for 90 min (HU). Mms21-5FLAG was immunoprecipitated and protein samples were analysed by immunoblotting. G) Mms21 interaction with Pol ε does not depend on S phase checkpoint components. Wild type cells and sml1Δ rad53Δ, mrc1Δ, tof1Δ, ctf18Δ and dcc1Δ mutants, carrying a tagged version of Dpb2, were arrested in G1 and synchronously released either in YPD for 30 min (S) or in medium containing 0.2 M HU for 90 min (HU). Dpb2-TAP was immunoprecipitated and protein samples were analysed by immunoblotting.
Fig 5
Fig 5. Pol2 is SUMOylated at K571.
A) Mms21 SUMOylates Pol2 N-terminal half. (Top). A graphic representation of the Pol2 allele used in the experiment is shown. POL2 was tagged at the C-terminal with a 9MYC tag. In addition, a 3xTEV sequence was inserted at the position S1227. (Bottom). Wild type and mms21-CH mutant cells, carrying the POL2-(3TEV)-9MYC allele, were grown to exponential phase, arrested in G1 and synchronously released in YPD 0.2 M HU for 90 min. Pol2 was then immunoprecipitated, incubated in the presence or absence of the 20 units of TEV protease, eluted by boiling and analysed by immunoblotting. The N-terminal part of Pol2 was detected with a polyclonal antibody raised against the N-terminal half of Pol2, while the C-terminal half was examined with an anti-MYC antibody. B) Mass spectrometry analysis reveals a single putative site of SUMOylation at Lysine 571. (Left) Cells carrying a Dpb2 TAP-tagged allele were synchronously released in medium containing 0.2 M HU for 90 min. Pol ε was immunoprecipitated at 700 mM potassium acetate and eluted by TEV cleavage. Samples were analysed by electrophoresis and stained in Coomassie blue. The Pol2-SUMO band was cut and digested with trypsin for mass spectrometry analysis. C) MS/MS spectrum showing Pol2 modification at K571 by SUMO. D) Pol2 SUMOylation is abolished in a K571R mutant. Cells carrying a DPB2 TAP-tagged allele and a wild type or a pol2 K571R (pol2KR) allele were grown to exponential phase, arrested in G1 and synchronously released either in fresh medium for 30 min (S) or in YPD 0.2 M HU for 90 min (HU). Dpb2-TAP was immunoprecipitated and analysed by immunoblotting. E) K571 is in the palm domain of Pol2. Illustration of the position of K571 within Pol2 N-terminal crystal structure (81). K571 is in a Pol2-specific large insertion within the palm domain.
Fig 6
Fig 6. Pol2 C-terminal contains a SUMO-interacting motif (SIM).
A) The C-terminal half of Pol2 interacts with SUMO. The ability of the subunits of Pol ε- Dpb2, Dpb3, Dpb4, Pol2 N-terminal (1–1265) and Pol2 (1128–2222)—and of Mrc1 to interact with SUMO (Smt3AAΔ, an allele that cannot be used as a moiety for SUMOylation) was tested by using the yeast two-hybrids assay. Pol2 C-terminal shows the ability to interact with SUMO. B) Mapping of the interaction between several fragments of Pol2 C-terminal half and SUMO was tested by yeast two-hybrids assay. Pol2 (2013–2222) fragment is sufficient for the interaction with SUMO, while the deletion of the last 30 amino acids blocks the binding. C) Identification of a SIM motif at the extreme C-terminal of Pol2. The analysis of the sequence identified a putative SIM sequence at the extreme C-terminal. Mutation of this sequence abolished the binding of the SUMO protein while not affecting the interaction with Dpb2. D) Pol ε composition and SUMOylation is not affected by the mutation of the SIM motif at the C-terminal. An untagged strain, POL2 or pol2sim cells carrying a TAP-tagged allele of DPB2, were grown to the exponential phase, arrested in G1 and synchronously released in YPD 0.2 M HU for 90 min. Dpb2-TAP was immunoprecipitated and protein samples were analysed by immunoblotting. E) Possible models of action of Pol2 SUMOylation in response to replication stress. Rad53, activated through the S phase checkpoint, and the E3 SUMO-ligase Mms21, interacting with the Pol ε, promote the SUMOylation of Pol2 at Lysine 571. This SUMOylation might then lead to (top) an intramolecular binding of SUMO by Pol2 SIM (or dimerization), (middle) recruitment of a SUMOylated factor at forks, or (bottom) recruitment of different proteins by the SUMO and SIM sequences.
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References

    1. Kotsantis P, Petermann E, Boulton SJ. Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place. Cancer Discov. 2018;8(5):537–55. 10.1158/2159-8290.CD-17-1461 - DOI - PMC - PubMed
    1. Gaillard H, García-Muse T, Aguilera A. Replication stress and cancer. Nat Rev Cancer. 2015;15(5):276–89. 10.1038/nrc3916 - DOI - PubMed
    1. Heller R, C., Kang S, Lam W, M., Chen S, Chan C, S., Bell S, P. Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. Cell. 2011;146(1):80–91. 10.1016/j.cell.2011.06.012 - DOI - PMC - PubMed
    1. Sheu YJ, Stillman B. Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol Cell. 2006;24(1):101–13. 10.1016/j.molcel.2006.07.033 - DOI - PMC - PubMed
    1. Tanaka S, Nakato R, Katou Y, Shirahige K, Araki H. Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. Curr Biol. 2011;21(24):2055–63. 10.1016/j.cub.2011.11.038 - DOI - PubMed

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