1. Volume 66, Issue 5, Pages 581-596.e6 (June 2017)
SUMO-Targeted DNA Translocase Rrp2 Protects the Genome from Top2-Induced DNA Damage 
Yi Wei, Li-Xue Diao, Shan Lu, Hai-Tao Wang, Fang Suo, Meng-Qiu Dong, Li-Lin Du 
Molecular Cell 
Volume 66, Issue 5, Pages e6 (June 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 66, 581-596.e6DOI: (10.1016/j.molcel.2017.04.017)
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3. Figure 1
rrp2Δ Mutant Exhibited an ETOP Sensitivity that Can Be Suppressed by SUMOylation-Related Mutations
(A) Schematic of barcode-sequencing-based ETOP sensitivity screen of the fission yeast deletion library in the pmd1Δ background.
(B) A scatterplot depicting ETOP sensitivity screen data. See Table S1 for the growth inhibition score of each mutant. Higher scores indicate stronger ETOP sensitivity. Genes within 500 kb of the pmd1 gene are removed to account for the linkage effect.
(C) An enrichment plot of a gene set enrichment analysis (GSEA) showing that genes in the gene set RESPONSE_TO_DNA_DAMAGE_STIMULUS are enriched among the ETOP-sensitive mutants (normalized enrichment score [NES] = 1.83, FDR q value = 0.013).
(D) ETOP, but not IR or CPT, caused more pronounced Rad52 focus formation in pmd1Δ rrp2Δ compared to pmd1Δ cells. Cells were treated with 36 Gy IR, 10 μM CPT, or 50 μg/mL ETOP. Values are means of triplicates ± SD. ETOP caused pmd1Δ rrp2Δ cells to elongate, presumably due to cell-cycle arrest. Scale bar, 5 μm.
(E) rrp2Δ enhanced the ETOP sensitivity of MRN and Ctp1 mutants.
(F) Suppressors of the ETOP sensitivity of rrp2Δ.
(G) pli1Δ, nup132Δ, and cdc48-A439T suppressed ETOP-induced Rad52 focus formation in pmd1Δ rrp2Δ cells. Values are means of triplicates ± SD.
See also Figure S1 and Table S1.
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4. Figure 2 Rrp2 Binds to SUMOylated Top2 Using SIMs
(A) Schematic showing the SUMOylation sites on Top2. Blue ovals, red ovals, and ovals colored half blue and half red denote sites identified only by Køhler et al. (Køhler et al., 2015), sites identified only in this work, and sites identified in both works, respectively (see Table S2 for a detailed list of sites identified in this work). The catalytic residue Y835 is marked by a triangle.
(B) Pli1 catalyzed in vitro SUMOylation of Top2. Mature SUMO (Pmt3GG) and SUMOylation enzymes were purified from E. coli. The substrate, YFP-FLAG-His6 (YFH)-tagged Top2, was expressed under the Pnmt1 promoter and immunopurified from S. pombe. Reactions were performed at 30°C for 10 min.
(C) ETOP did not affect Top2 SUMOylation. ETOP caused the formation of covalent Top2-DNA complex (Top2cc), which has a low mobility on SDS-PAGE and is sensitive to DNase. A small amount of Top2cc formed without DNA addition (lane 3), probably due to DNA contamination in the Top2 preparation.
(D) Fusing Ulp1( ) to the C terminus of Top2 following mCherry (Top2-mCherry-Ulp1) rescued the ETOP sensitivity of pmd1Δ rrp2Δ. A Ulp1 catalytically inactive mutation (C527S, Top2-mCherry-Ulp1∗) abolished this rescue effect.
(E) Rrp2 co-immunoprecipitated SUMOylated Top2. Top2 was endogenously tagged with 3×HA. GFP or GFP-Rrp2 was expressed under the rrp2 promoter. A strain in which SUMO is tagged with a His10 tag was used to determine whether the up-shifted bands of Top2 were its SUMOylated forms.
(F) GST pull-down assay showing that the N-terminal 360 amino acids of Rrp2 are responsible for Top2-SUMO conjugate binding. Left panel, GST and GST-tagged Rrp2 proteins used for the pull-down assay. Right panel, pull-down of in vitro SUMOylated Top2.
(G) SUMO-interacting motifs (SIMs) in Rrp2. Upper panel depicts the domain organization of Rrp2. Middle panel depicts the SIM∗ mutations that substitute hydrophobic residues with alanines. Lower panel shows the alignments of SIM3 and SIM5.
(H) SIMs in Rrp2 are important for SUMO chain binding. Upper panel, GST pull-down of SUMO chains. Asterisks denote nonspecific bands. Lower panel, GST and GST-tagged proteins used for the pull-down assay.
(I) SIMs in Rrp2 are important for its ability to confer ETOP resistance. Different versions of Rrp2 were expressed under the rrp2 promoter in pmd1Δ rrp2Δ. Expression levels are shown in Figure S2F.
See also Figure S2 and Table S2.
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5. Figure 3
Rrp2 Prevents the Degradation of Chromatin-Bound Top2-SUMO Conjugates
(A) Higher molecular weight Top2-SUMO conjugates decreased in rrp2Δ. Endogenously TAP-tagged Top2 was immunoprecipiated using IgG beads and immunoblotted using an anti-SUMO antibody, which is also reactive with the TAP tag.
(B) ETOP treatment did not notably affect Top2-SUMO conjugates. Cells were treated with 50 μg/mL ETOP for the indicated time.
(C) Chromatin-bound Top2 decreased in rrp2Δ. Histone H3 served as loading control.
(D) pli1Δ and cdc48-A439T reversed the decrease of chromatin-bound Top2 in rrp2Δ.
(E) Proteasome inhibitor BTZ reduced ETOP-induced, but not zeocin-induced, Rad52-YFP foci in rrp2Δ cells of the MDR-supML background. Cells were treated with 2 μg/mL ETOP or 5 μg/mL zeocin alone or in combination with 5 μM BTZ or 10 μg/mL MBC for 2 hr. Values are means of triplicates ± SD. Representative images are shown in Figure S3C.
(F) Diagram depicting the lacO-LacI system used to concentrate Top2 at a specific genomic locus.
(G) Top2-LacI targeted to the lacO repeat exhibited diminished focus formation in rrp2Δ and caused local DNA damage in a manner dependent on the catalytic residue Y835. Values are means of triplicates ± SD.
(H) Top2 ubiquitination increased in rrp2Δ. Biotin-tagged ubiquitin (Biotin-Ub) was expressed from the Pnmt1 promoter and was immunoprecipitated with streptavidin beads. cdc cells were shifted to 37°C for 2 hr to inactivate Cdc48.
(I) pli1Δ diminished Top2 ubiquitination in rrp2Δ.
Except for (E), strains used in this figure are in the pmd1Δ background, and ETOP treatment was performed by incubating with 50 μg/mL ETOP for 2 hr.
See also Figure S3.
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6. Figure 4
Top2-4×SUMO Is a STUbL Substrate, and a Partial Loss-of-Function STUbL Mutation Renders Rrp2 Dispensable for ETOP Resistance
(A) Top2-4×SUMO was ubiquitinated by Rfp1/2-Slx8 STUbL in a manner dependent on the SUMO chain fused to Top2 and the SIMs on STUbL. Ubiquitin and ubiquitination enzymes were purified from E. coli. The substrate, Top2-4×SUMO or Top2, was expressed under the Pnmt1 promoter and immunopurified from S. pombe. Reactions were incubated at 30°C for 10 min.
(B) GST pull-down assay showing that Rfp1 and Rfp2, but not Rfp1-2×SIM∗, can bind to SUMO chains.
(C) STUbL-mediated Top2-4×SUMO ubiquitination was stimulated by DNA and ETOP.
(D) slx8-R247G rescued the ETOP sensitivity of pmd1Δ rrp2Δ.
(E) Slx8-R247G mutation weakened the STUbL activity of Rfp1-Slx8 toward GST-4×SUMO.
(F) The E3 activity of Slx8-R247G-containing STUbL toward Top2-4×SUMO was inhibited by DNA and ETOP. Reactions were carried out as in (A) and (C), except that the reaction time was 30 min instead of 10 min.
(G) Slx8-R247G mutation disrupted STUbL-DNA binding. Proteins bound to biotinylated double-stranded DNA were pulled down by streptavidin beads and analyzed by immunoblotting with HA antibody.
(H) In vitro ubiquitination of an artificial substrate, GST-4×SUMO-LacI, by Rfp1-Slx8 was influenced by lacO repeat DNA and IPTG.
See also Figure S4.
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7. Figure 5
Rrp2 Uses Its ATPase Activity to Displace SUMOylated Top2 from DNA
(A) Mutating the ATPase motifs of Rrp2 abolished its ability to confer ETOP resistance. N-terminally GFP-tagged Rrp2 proteins were expressed under the rrp2 promoter in pmd1Δ rrp2Δ. Expression levels are shown in Figure S5E.
(B) The level of chromatin-bound Top2 was higher in slx8-29 rrp2Δ than in slx8-29. slx8-29 mutants were grown at 25°C to mid-log phase and then shifted to 35°C for 5 hr.
(C) Mutating SIMs or an ATPase motif rendered Rrp2 unable to decrease the chromatin-bound Top2 in slx8-29 rrp2Δ.
(D) The level of SUMOylated Top2 was higher in slx8-29 rrp2Δ than in slx8-29.
(E) Rrp2 can displace SUMOylated Top2 from DNA in a dose-dependent manner. Top2-Y835F bound to DNA-coated beads was left unmodified or was SUMOylated and then incubated with Rrp2 in the presence of 5 mM ATP. Rrp2 was immunopurified from S. pombe.
(F) The displacement activity of Rrp2 toward SUMOylated Top2 requires SIMs and the ATPase activity.
(G) Diagram depicting the in vitro Top2cc formation assay.
(H) Rrp2 reduced Top2cc formed by Top2-4×SUMO.
(I) Mutating ATPase motifs rendered Rrp2 unable to decrease Top2cc formed by Top2-4×SUMO.
(J) Mutating SIMs, but not the RING domain, rendered Rrp2 unable to decrease Top2cc formed by Top2-4×SUMO.
See also Figures S5 and S6.
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8. Figure 6
Rrp2 Antagonizes STUbL by Inhibiting Its Binding to SUMO Chains
(A) SIMs in Rrp2 remained indispensable even when Rrp2 was tethered to Top2 using the GBP-GFP interaction.
(B) Rrp2 can compete with STUbL for Top2-4×SUMO binding in a SIM-dependent manner. Biotin-tagged Top2-4×SUMO was bound to streptavidin beads and incubated with STUbL proteins and increasing amount of FLAG2-His6 (FFH)-tagged Rrp2 immunopurified from S. pombe.
(C) Rrp2-N360 can compete with Rfp1 and Rfp2 for SUMO chain binding. GST-Rfp1 or Rfp2 immobilized on glutathione beads was first allowed to bind recombinant SUMO chains and then incubated with increasing amount of Rrp2-N360 purified from bacteria.
(D) Rrp2 can inhibit Top2-4×SUMO ubiquitination by STUbL.
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9. Figure 7
Budding Yeast Uls1 Confers ETOP Resistance in a Manner Similar to Rrp2
(A) uls1Δ cells exhibited ETOP sensitivity. ULS1 was deleted in a regular lab strain background and two drug-hypersensitive backgrounds (Chinen et al., 2011; Stepanov et al., 2008).
(B) ETOP, but not IR or CPT, caused more pronounced Rad52 focus formation in uls1Δ compared to ULS1 cells. Cells were treated with 64 Gy IR, 3 μM CPT, or 10 μg/mL ETOP. Values are means of triplicates ± SD. Scale bar, 5 μm.
(C) uls1Δ enhanced the ETOP sensitivity of rad52Δ.
(D) The SIM-containing N-terminal region and ATPase motifs are important for the ability of Uls1 to confer ETOP resistance.
(E) Deleting SIZ1 and SIZ2 suppressed the ETOP sensitivity of uls1Δ.
(F) A model of how Rrp2 prevents STUbL-dependent Top2cc degradation. Rrp2 displaces SUMOylated Top2 from chromatin to reduce the chance of Top2cc becoming a substrate of STUbL. Rrp2 also antagonizes STUbL by inhibiting its SUMO chain binding.
See also Figure S7.
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