1. Volume 68, Issue 4, Pages 758-772.e4 (November 2017)
Cohesin Ubiquitylation and Mobilization Facilitate Stalled Replication Fork Dynamics 
Camilla Frattini, Sara Villa-Hernández, Grazia Pellicanò, Rachel Jossen, Yuki Katou, Katsuhiko Shirahige, Rodrigo Bermejo 
Molecular Cell 
Volume 68, Issue 4, Pages e4 (November 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 68, 758-772.e4DOI: (10.1016/j.molcel.2017.10.012)
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3. Figure 1
Rsp5Bul2-Mediated Ubiquitylation Promotes Survival to Replication Stress
(A) Rsp5-associated factors. Serial dilutions of wild-type (WT) and bul2Δ cells on yeast peptone dextrose adenine (YPDA) (−) or in the presence of 200 mM HU or 0.02% MMS are shown.
(B–D) Serial dilutions of wild-type, rsp5-1, rsp5-25, bul1Δ, bul2Δ, and bul1Δ bul2Δ cells (B); Rsp5 adaptor mutants (C); and wild-type, ubp2Δ, bul2Δ, bul2Δ ubp2Δ, bul1Δ bul2Δ, and bul1Δ bul2Δ ubp2Δ cells (D).
(E and F) Fluorescence-activated cell sorting (FACS) analysis of logarithmically growing (Log) wild-type and bul1Δ bul2Δ cells released from a G1 block (αF) into S-phase in the presence of 200 mM HU (E) or in fresh medium (F). Merged profiles from HU-treated cells are shown.
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4. Figure 2
Rsp5Bul2-Mediated Ubiquitylation Promotes Stable Stalled Fork Progression
(A) 2D gels in wild-type and bul1Δ bul2Δ cells released from G1 in the presence of 200 mM HU. Histograms show quantified replication intermediates. Restriction strategy, probes, and scheme of 2D gel replication intermediate profiles are shown.
(B) CGS in wild-type and bul1Δ bul2Δ cells. Red histogram bars show S-phase to G1 log10 read ratios higher than 1 (10°) along a region on chromosome I. Asterisks mark a repetitive region rendering uninformative ratios.
(C) SCE rates in wild-type and bul1Δ bul2Δ cells grown in the presence or absence of 20 mM HU. Mean/SDs of three independent experiments are shown.
(D) SCE rates in wild-type, bul2Δ, ubp2Δ, and bul2Δ ubp2Δ cells grown in the presence or absence of 75 mM HU.
See also Data S1.
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5. Figure 3
Rsp5Bul2 Physically Interacts with the Mec1/Ddc2 Checkpoint Kinase and Cohesin Complexes
(A) Comassie staining of IP fractions from S-synchronized cells. Asterisks mark excised bands.
(B) CoIP analysis in cells expressing epitope-tagged versions of Ddc2, Rsp5, and Bul2 released into S-phase in the presence of 200 mM HU. no Ab, IP controls without antibodies; WCE, whole-cell extract; SUP, supernatant.
(C) CoIP analysis of Bul2 and Smc1/Smc3 in cells released into S-phase in the presence of 200 mM HU. Structural arrangement of cohesin subunits is shown.
See also Figures S1 and S2.
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6. Figure 4
Cohesin Is Ubiquitylated in an Rsp5Bul2- and Checkpoint-Dependent Manner
(A and B) Ubiquitylation assays in Smc1-PK, Smc3-PK, Scc1-HA, or Scc3-Myc cells carrying YEplac195-CUP1 (−) or YEplac195-CUP1-HIS7-UB (UbHIS) vectors released into S-phase in the presence of 200 mM HU using a regular (A) or stringent (B) protocol. The dashed lines indicate the position of lanes removed from the original blot.
(C) Ubiquitylation assays in HU-treated wild-type cells or rsp5-25 and bul1Δ bul2Δ mutants.
(D) Ubiquitylation assays in HU-treated MEC1 or mec1-100 cells.
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7. Figure 5
Rsp5Bul2-Mediated Ubiquitylation Stimulates Cohesin Function in Fork Protection
(A) Serial dilutions of wild-type, smc3-42, smc1-259, bul1Δ bul2Δ, smc3-42 bul1Δ bul2Δ, and smc1-259 bul1Δ bul2Δ cells.
(B) FACS of wild-type, bul1Δ bul2Δ, smc1-259, and bul1Δ bul2Δ smc1-259 cells released from G1 (αF) into S-phase in the presence of 200 mM HU.
(C) Serial dilutions of wild-type, ubp2Δ, smc3-42, smc3-42 ubp2Δ, smc1-259, and smc1-259 ubp2Δ cells.
See also Figures S3 and S4.
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8. Figure 6
Rsp5Bul2-Dependent Ubiquitylation and Targeting by Cdc48 Promote Cohesin Mobilization from Chromatin and Replication Stress Survival
(A) Serial dilutions of wild-type, cdc48-3, bul1Δ bul2Δ, and cdc48-3 bul1Δ bul2Δ cells.
(B) Serial dilutions of wild-type, ubp2Δ, cdc48-3, and cdc48-3 ubp2Δ cells.
(C) Serial dilutions of wild-type, cdc48-3, smc1-259, cdc48-3 smc1-259, smc3-42, and cdc48-3 smc3-42 cells.
(D) ChIP-qPCR of Smc1-PK and Mcm7-HA binding in wild-type (BUL1 BUL2) cells or bul1Δ bul2Δ mutants released from G1 into 200 mM HU. Mean and SDs of three independent experiments are shown.
(E) ChIP-qPCR of Smc1-PK binding close to ARS305 in wild-type (CDC48) cells or cdc48-3 mutants upon release from G1 into 200 mM HU.
(F and G) ChIP-qPCR of Smc1-PK binding close to ARS306 (F) and ARS607 (G) in wild-type cells and bul1Δ bul2Δ or cdc48-3 mutants (grown at 30°C or 32°C, respectively) released from G1 into 200 mM HU.
(H) Serial dilutions of wild-type, rad61Δ, cdc48-3, and rad61Δ cdc48-3 cells.
(I) Serial dilutions of wild-type, GAL1-RAD61, cdc48-3, and cdc48-3 GAL1-RAD61 cells on yeast peptone (YP) raffinose containing (+GAL) or not (−GAL) galactose.
See also Figure S5.
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9. Figure 7
Cohesin Mobilization Facilitates Nascent Sister Chromatid Entrapment and Protection of Stalled Forks
(A) Schematic of nascent strand-binding assay (NSBA, green) and ChIP (red) qPCR procedure. Shaded DNA fragments are present in the respective INPUT, but not IP, fractions. Dotted-line boxes mark regions amplified by qPCR. Smc1 NSBA performed in CDC48 and cdc48-3 cells expressing Smc1-PK released from G1 into 200 mM HU, performed on BrdU-labeled (BrdU) and unlabelled (−) cells at ARS305, and a cohesin-bound control (Ctrl) region are shown. Mean and SDs of three independent experiments are shown.
(B) Smc1 NSBA in BUL1 BUL2 (wild-type) and bul1 bul2 cells expressing Smc1-PK upon release from a G1 block into 200 mM HU for 1 hr.
(C) Sister chromatid cohesion assay with GFP-marked URA3 locus (wild-type, smc1-259, bul1Δ bul2Δ, cdc48-3, and rad61Δ cells).
(D) A model for dynamic interfacing of cohesin and stalled replication forks. Mec1 promotes cohesin ubiquitylation and cohesin is engaged by Cdc48, resulting in its dislodgement from chromatin. Cdc48 may remodel cohesin structure assisted by Wapl. Mobilization enables cohesin re-positioning behind the stalled replisome, poised for entrapment of stalled nascent chromatids. Eco1 locks cohesin DNA exit gate by acetylation of Smc3, thus fastening nascent strand-replisome architecture and protecting fork functionality. DNA synthesis and fork progression can resume upon checkpoint-mediated upregulation of dNTP pools.
See also Figures S6 and S7.
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