1. Volume 49, Issue 3, Pages 536-546 (February 2013)
Noncanonical Role of the Clamp in the Error-Free DNA Damage Tolerance Pathway 
Georgios Ioannis Karras, Marco Fumasoni, Grzegorz Sienski, Fabio Vanoli, Dana Branzei, Stefan Jentsch 
Molecular Cell 
Volume 49, Issue 3, Pages (February 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 49, 536-546DOI: (10.1016/j.molcel.2012.11.016)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 9-1-1 Is Required for Error-Free DDT
(A) Screen for mutants that are defective in the error-free RAD6 pathway. The error-free RAD6 pathway involves polyubiquitylated PCNA and SCJs as intermediates (central diagrams). In pol32Δ sgs1Δ double mutants (which experience replication stress), toxic recombination intermediates (SCJs) accumulate, causing a synthetic growth defect (upper row). In the mutants of the error-free RAD6 pathway, the growth defect is suppressed (lower row). Lower scheme: Outline of a robot-assisted screen for genes involved in the error-free RAD6 pathway. An sgs1Δ pol32Δ double mutant harboring a URA3 gene-containing pCEN-SGS1 plasmid was mated to a library of viable deletion mutants (see Supplemental Experimental Procedures for details). Cells contain the pCEN-SGS1 (URA3) plasmid on plates lacking uracil, but are selected against this plasmid on 5′-FOA plates. Thus, in the final genetic background (pol32Δ sgs1Δ), only mutants that are defective in the error-free RAD6 pathway survive.
(B) Loss of subunits or the Rad24 clamp loader suppresses the growth defect of sgs1Δ pol32Δ double mutants. As in (A), selection against pCEN-SGS1 (URA3) on 5′-FOA plates reveals a growth defect of pol32Δ sgs1Δ cells that can be suppressed by mutants deficient in PCNA polyubiquitylation (mms2Δ) or (rad17Δ or ddc1Δ).
(C) Loss of subunits or the Rad24 clamp loader suppresses the cold sensitivity of pol32Δ. Spotting of 1:5 serial dilutions of cultures on YPD plates incubated at 30°C for 2.5 days or 17°C for 4 days is shown. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Dual Role of 9-1-1 in Error-Free DDT
(A) SCJ accumulation is linked to early and late in the cell cycle. Cycling (cycl) strains sgs1Δ, sgs1Δ ubc13Δ, sgs1Δ rad17Δ, and sgs1Δ rad17Δ ubc13Δ were synchronized in G1 with α-factor and then released in YPD media containing MMS 0.033% v/v. Samples for 2D gel and FACS analysis were taken at various time points. The highest value (sgs1Δ at 240 min) obtained during quantification of X molecules (SCJs; arrowheads) was put to 100% for the quantification shown below.
(B) Suppression of the rad18Δ MMS sensitivity by srs2Δ depends on WT and indicated mutant cells were spotted on YPD plates containing different MMS concentrations and incubated at 28°C for 3 days. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Interplay between 9-1-1 and PCNA during DDT
(A) PCNA polyubiquitylation enhances replication stress-induced G2/M checkpoint signaling. Replication stress is induced in pol32Δ, and PCNA polyubiquitylation is absent in ubc13Δ. Exponentially growing cultures were arrested in G1 with α-factor, released into the cell cycle by removing α-factor by washing and pronase treatment, and grown at 14°C. At the indicated time points, samples were collected and analyzed by FACS (lower panel) and western blot using antibodies specific for PCNA, Clb2, and Rad53. The Pgk1 level served as the loading control. Modified species of PCNA (U1, monoubiquitylated; U2, diubiquitylated; and SK164, SUMOylated at K164) are indicated. The asterisk denotes a cross-reacting band. The numbers below the anti-Rad53 panel give the percentage of phosphorylated Rad53 of the total pool of Rad53.
(B and C) Influence of PCNA polyubiquitylation on chromatin binding during different cell-cycle phases upon replication stress. Exponentially growing cells were synchronized as in (A), and samples for chromatin-binding analysis were collected after 3 hr (B, S phase) or 6 hr (C, G2/M phase) of growth at 14°C. Samples from the indicated extracts (WCE, whole-cell extract; SUP, supernatant; CHR, solubilized by DNase chromatin fraction; and HS, solubilized by high-salt treatment of the remaining insoluble fraction, which is enriched for histone marks such as H3K4me3) were analyzed by western blot using the indicated antibodies. A blot for the cytosolic protein Dpm1 is shown as control. Quantifications of the subunit Mec3 signal for each strain (normalized against H2A levels) are plotted below each experiment. One representative experiment is shown from three different examples. AU, arbitrary units.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
9-1-1-Dependent Error-Free DDT Pathways Are Functional in G2/M
(A) G2-RAD17 cells are defective in S phase checkpoint signaling upon replication stress. Cells were synchronized as in Figure 3A. Cell-cycle progression was followed by FACS analysis (bottom panel) and western blots (upper panels) using antibodies against Clb2 and Rad53. Pgk1 signals serve as loading control. Top left: G2-RAD17 expression parallels the expression of Clb2. Top right: pol32Δ cells experience replication stress, which results in S phase delay (see late emergence of the 2N peak) and Rad53 phosphorylation at later time points (G2/M). Bottom left: S phase is significantly shorter in cells defective in (rad17Δ; see early emergence of the 2N peak). Bottom right: Expression of G2-RAD17 restores Rad53 phosphorylation in pol32Δ in G2/M, but does not restore the longer S phase of pol32Δ cells (expressing WT RAD17). Note that the onset of Rad53 phosphorylation occurs earlier in G2-RAD17 pol32Δ cells as compared with pol32Δ cells, due to the shorter S phase duration. A plot of phosphorylated Rad53 quantification is shown in the bottom panel.
(B) G2/M-restricted G2-Rad17 or WT RAD17 is required for the cold sensitivity of pol32Δ cells and supports WT resistance to MMS-induced DNA lesions. Spotting of 1:5 serial dilutions on YPD plates supplemented with the indicated MMS concentrations and grown at 30°C for 2.5 days, or incubated at 17°C for 4 days, is shown. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 9-1-1 Operates in Error-Free DDT Independently of Rad9
(A and B) The indicated diploid strains were dissected, and haploid spores were grown at 30°C for 3 days. Spores derived from the same tetrads are presented in vertical configuration. Deletions of RAD24 (A) or MMS2 (B) suppress the lethality of pol32Δ rad9Δ cells.
(C) Schematic description of a robot-assisted screen for deletion mutant suppressors of rad9Δ pol32Δ’s synthetic lethality.
(D) Deletion of EXO1 suppresses the synthetic lethality of rad9Δ pol32Δ.
(E) Deletion of EXO1 or mutation of its nuclease active site suppresses the cold sensitivity of pol32Δ. Strains bearing the indicated plasmid (right) were spotted on SC plates lacking leucine containing MMS or no drug and were incubated at the indicated temperature.
(F) Increase in spontaneous mutagenesis at the CAN1 locus by deletion of EXO1 is comparable and epistatic to deletion of MMS2. Averages and standard deviations were obtained from three independent experiments.
(G) Expression levels of WT Exo1 and mutants expressed exogenously in cells lacking EXO1 were detected using an anti-Exo1 antibody. Pgk1 was used as loading control.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Model for the Roles of 9-1-1 in Error-Free DDT Pathways
(A) Replication of damaged DNA results in ssDNA gaps containing the DNA lesions.
(B) is loaded on the 5′ ends of ssDNA gaps and facilitates S phase elongation and stabilization of proximal replication forks.
(C and C′) In parallel, template switching is initiated at the 3′ ends as a result of the concerted action of polyubiquitylated PCNA and recombination proteins (Rad51). Crucial components of each error-free DDT pathway are highlighted. Exo1 may expand the sizes of the ssDNA gaps promotes RAD6-mediated error-free DDT during S phase (C). In G2/M, is required for a fraction of the HR pathway, which is kept in check by SUMOylated PCNA and Srs2 during S phase (C’). Delaying error-free ssDNA gap filling (e.g., by Polδ defects arising in pol32Δ cells) may result in increased gap size and robust activation of the G2/M checkpoint via recruitment of Rad9.
(D) Completion of template switching results in the formation of SCJs (detectable by 2D gel electrophoresis) that are subsequently dissolved by the Sgs1/Top3 complex. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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