SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks

Abstract Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.

A. Sensitivity of indicated strains to indicated genotoxic drugs.Ten-fold serial dilutions of exponential cultures were dropped on appropriate plates.Bleo: bleomycin; CPT: camptothecin; HU: hydroxyurea; MMS: methyl methane sulfonate.
B. Flow cytometry analysis of indicated strains in indicated conditions.Logarithmically growing cells (AS: Asynchronous cells) were exposed to 20 mM HU for 4 hours (HU time point) and then released into fresh, HU-free, rich medium YES at 30 o C to monitor S-phase progression at the indicated time after release.Arrows indicate the delay in S phase progression compared to WT.
C. Analysis of chromosomes by pulse field gel electrophoresis (PFGE) in the above-mentioned conditions (as in b).Representative images of chromosome migration during PFGE in indicated strains and conditions.D. Quantification of % of chromosomes migrating into the gel after release from HU block.Values are means of two independent biological replicates ± standard deviation (SD).p value was calculated by two-sided Fisher's exact test (**** p ≤0.0001).C Expression of Ulp1-GFP in indicated strains and conditions.An untagged WT strain was included as a control for antibody specificity.Tubulin was used as a loading control.The binding of Npp106-GFP to the RFB was analysed by chromatin immuno-precipitation (ChIP) followed by qPCR analysis using either anti-GFP antibody or IgG as control in indicated strains and conditions.Starting quantities were normalized to a control and unrelated locus and to Wild type RFB Off condition, in the absence of tethering.Values are the mean of 3 to 4 independent biological replicates, errors bars correspond to stander error of the mean (sem).Statistical difference was detected between the samples using the two-sided t-test.* p<0.05.

Figure S1 :
Figure S1: Role of the nuclear basket in the recovery from HU-induced stalled forks.

Figure S2 :
Figure S2: The downregulation of Ulp1 expression is caused by the proteasome.A. Ulp1-GFP is a functional fusion protein.Ten-fold serial dilutions of exponential cultures were dropped on appropriate plates.Bleo: bleomycin; CPT: camptothecin; HU: hydroxyurea; MMS: methyl methane sulfonate.B. Cell imaging of Ulp1-GFP in indicated strains and conditions.Representative cell images of Ulp1-GFP in indicated strains in presence or absence of bortezomib.Scale bar: 5µm.

Figure S3 :
Figure S3: Image quantification of nuclear morphology parameters, Ulp1-GFP and Cut11-mCherry intensity.A. Example of fluorescence images of nuclei in cells expressing the endogenous Cut11-mCherry fusion protein in indicated strains.Scale bare 2 µm.B. Box-and-whisker plots of nucleus perimeter (left panel) and nucleus size (right panel) in indicated strains.Boxes represent the 25/75 percentile, black lines indicate the median, the whiskers indicate the 5/95 percentile and dots correspond to minimum and maximum values.p value was calculated by Mann-Whitney U test (**** p ≤0.0001; ns: non-significant).At least 50 nuclei were analyzed for each strain.C. Box-and-whisker plots of Ulp1-GFP total intensity (raw integrated density) at the nuclear periphery in indicated strains and conditions.Boxes represent the 25/75 percentile, black lines indicate the median, the whiskers indicate the 5/95 percentile and dots correspond to minimum and maximum values.p value was calculated by Mann-Whitney U test (**** p ≤0.0001; * p≤0.05; ns: non-significant).At least 50 nuclei were analyzed for each strain.D. Box-and-whisker plots of Cut11-mCherry total intensity (raw integrated density) at the nuclear periphery in indicated strains and conditions.Boxes represent the 25/75 percentile, black lines indicate the median, the whiskers indicate the 5/95 percentile and dots correspond to minimum and maximum values.p value was calculated by Mann-Whitney U test (**** p ≤0.0001; *** p≤0.001; ** p≤0.01; ns: non-significant).At least 50 nuclei were analyzed for each strain.E. Box-and-whisker plots of co-localization between Cut11-mCherry and Ulp1-GFP (using the Pearson correlation coefficient) in indicated strains.The co-localization between the Npp106-GFP, an inner ring nucleoporin of NPC, and Cut11-mCherry, was performed as a control to show maximum correlation between intensities of both proteins at the resolution achieved on the images.Boxes represent the 25/75 percentile, black lines indicate the median, the whiskers indicate the 5/95 percentile and dots correspond to minimum and maximum values.p value was calculated by Mann-Whitney U test (**** p ≤0.0001; ns: non-significant).At least 50 nuclei were analyzed for each strain.

Figure S4 :
Figure S4: An auxin-induced degron approach to conditionally downregulate Nup211.A. Expression of Nup211-HA-mAID fusion protein in strains expressing TIR1-F74A (right panels) or not (left panels) as a function of time (in minute) upon addition of 5-adamentyl-IAA.PCNA was used as a loading control.B. Top panels: expression of Ulp1-GFP, Nup211-HA-mAID and TIR1 in indicated conditions.PCNA was used as loading control.Bottom panel: quantification.Dots represent values obtained from independent biological experiments.The normalized amount of Ulp1 was calculated by dividing the GFP signal by PCNA.The normalized amount of Ulp1-GFP in mutants was indicated as a percentage of the WT.p value was calculated by two-sided Fisher's exact test (**** p≤0.0001; ** p≤0.01).C. Cell growth assay of indicated strains.Ten-fold serial dilutions of exponential cultures were dropped on plates containing 5-adamantyl-IAA (right panel) or not (left panel).Five independent clones expressing Nup211-HA-mAID were tested alongside the WT strain.Note the cell growth defect of Nup211-HA-mAID strains in the absence of 5-adamantyl-IAA is indicative of a lack of Nup211 functionality.D. Box-and-whisker plots of Ulp1-GFP density (mean fluorescence intensity) at the nuclear periphery in indicated strains and conditions.Boxes represent the 25/75 percentile, black lines indicate the median, the whiskers indicate the 5/95 percentile and dots correspond to minimum and maximum values.p value was calculated by Mann-Whitney U test (**** p ≤0.0001; ns: non-significant).At least 50 nuclei were analyzed for each strain.

Figure S5 :
Figure S5: Artificial targeting of Ulp1-LexA to LexA binding sites-containing RFB allows the tethering of the RFB to NPC in wild type and nup60 cells.

Figure S6 :
Figure S6: Characterization of rpn10 mutant.A. The absence of Rpn10 results in cell sensitivity to replication blocking agents.Ten-fold serial dilutions of exponential cultures were dropped on appropriate plates.HU: hydroxyurea; MMS: methyl methane sulfonate.B. Expression of SUMO conjugates in indicated strains.A strain deleted for pmt3 gene that encodes the SUMO polypeptide (sumo) was added as control for antibody specificity.* indicates unspecific signal.C. Top panel: scheme of replication intermediates (RI) analyzed by neutral-neutral 2DGE of the AseI restriction fragment in RFB OFF and ON conditions.Partial restriction digestion caused by psoralencrosslinks results in a secondary arc indicated on scheme by blue dashed lines.Bottom panels: representative RI analysis in indicated strains and conditions.The ura4 gene was used as a probe.The red arrow indicates the tail signal resulting from resected forks.D. Quantification of resected forks in indicated strains.Dots represent values obtained from independent biological experiments.Statistical difference was detected between the samples using the two-sided t-test.* p<0.05.E. Top panel: expression of Ulp1-GFP in indicated strains.An untagged WT strain was included as control for antibody specificity.Tubulin was used as a loading control.Bottom panel: quantification.The normalized amount of Ulp1 was calculated by dividing the GFP signal by tubulin signal.The normalized amount of Ulp1-GFP in the mutants is indicated as a percentage of the WT.Dots represent values obtained from independent biological experiments.F. Example of fluorescence (bottom panel) and bright-field images (top panel, DIC) of cells expressing the endogenous Ulp1-GFP fusion protein in indicated strains.Scale bare 5 µm.