1. PCNA Controls Establishment of Sister Chromatid Cohesion during S Phase 
George-Lucian Moldovan, Boris Pfander, Stefan Jentsch 
Molecular Cell 
Volume 23, Issue 5, Pages (September 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 PCNA Interacts Physically and Functionally with Eco1
(A) PCNA A251V mutants have cohesion defects. Quantification of cohesion defects of the indicated strains were assayed by using yeast strains GFP tagged on two different positions on ChrIV. Cells were arrested in metaphase as diploid, large-budded cells, and the number of GFP foci present in each cell was scored, indicating either normal cohesion (one signal) or a cohesion defect (two signals; see examples in micrographs). Each strain was assayed four to ten times, and for each experiment, at least 50 cells were counted. Error bars represent standard deviations.
(B) Eco1 interacts with PCNA in vivo. 3mycEco1 expressed from the ADH1 promoter was immunoprecipitated with anti-myc antibodies. Samples were analyzed by Western blot for myc or PCNA reactivity.
(C) Recombinant GST fusions of Eco1 and Chl1 were expressed and purified from E. coli (left) and used to pull down recombinant or endogenous PCNA (right panels) from yeast native extracts. Asterisks denote crossreacting proteins.
(D) Diagram showing Eco1 domains and the truncations used for GST pull-downs.
(E) GST pull-downs with recombinant Eco1 truncations and recombinant or endogenous PCNA.
(F) Mapping of the binding site of PCNA by two-hybrid analysis, using truncations of Eco1.
(G) Complementation analysis showing that the PCNA-interaction motif, the zinc finger, and the acetyl transferase (ACT) domain are all required for the essential function of Eco1. Different Eco1 truncations were assayed for their ability to support cell viability in the absence of wt ECO1, which is expressed from a plasmid that can be counter selected by plating the cells on 5-fluoro-orotic acid (5FOA)-containing medium.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Eco1 Mediates Establishment of Sister Chromatid Cohesion by Binding to PCNA
(A and B) The ability to interact with PCNA and the zinc finger are required for the essential function of Eco1. (A) Viabilities of eco1 mutants defective in the PCNA-interaction domain (marked with a light gray box) or in the zinc finger (dark gray box) were assayed by shuffling out wt ECO1 upon plating on 5FOA-containing medium.
(B) Two-hybrid analysis of the interaction of eco1 mutant proteins with PCNA, showing that the N terminus, but not the zinc finger, is required for this interaction.
(C–E) The PCNA-Eco1 interaction is required for efficient sister chromatid cohesion. (C) The mutant eco1S12A,K13A, partially defective in PCNA binding, shows cohesion defects. The assays for the cohesion defects of mutant eco1S12A,K13A, using GFP tagging of ChrIV was done as described in Figure 1A. ctf18Δ cells, impaired in establishment of cohesion (Mayer et al., 2001), were used as control. Each strain was assayed four to ten times, with 50 cells being counted in each assay. Error bars represent standard deviations. (D) An eco1 mutant, which expresses under control of its endogenous promoter a variant defective in PCNA binding (eco1Q18A,K20A,L21A) cannot complement the cohesion defects of eco1ctf eco1ctf7-203 cells with tagged ChrIV were arrested in G1 by α factor treatment and released into medium containing nocodazole at the restricted temperature (34°C) of the mutant. G2/M-arrested cells were microscopically assayed for cohesion defects. For each strain, three to six experiments were done in which at least 50 cells were counted. Error bars represent standard deviations. (E) Endogenous expression of wild-type Eco1, but not of eco1Q18A,K20A,L21A, can rescue the viability of eco1ctf7-203 mutants at restrictive temperatures.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The PCNA Binding Site Represents a Variant PIP Box
(A) A conserved PCNA-interaction motif is present in all Eco1 family members. Alignment of homologous sequences to S. cerevisiae Eco1 from residues 11 to 27. The positions occupied by the conserved amino acids Q and L/I, marked with an asterisk, match the core of the PCNA-interaction motifs PIP box and KA box. At the bottom, a diagram shows the domain organization of Eco1 family members.
(B and C) The Eco1 PIP box is required for interaction with PCNA in two-hybrid assays (B) and for cellular viability (C).
(D) ESCO2, a human homolog of Eco1, interacts with yeast (y) and human (h) PCNA.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Chromatin Association of Eco1 Depends in Part on PCNA Interaction
(A) Chromatin fractionation experiment using cells expressing 3myc-tagged wild-type Eco1, or mutants with defective PCNA interaction, reveal that mutations in the PCNA-interacting domain of Eco1 reduce its ability to bind chromatin. PCNA and H2B (controls) are chromatin associated, whereas the cytoplasmic Dpm1 protein was used as a negative control. 10% of the input, 100% of the pellet, and 5% of the supernatant fractions were loaded.
(B) Quantification of chromatin-association activity of Eco1 variants. Bars represent the percentage of chromatin bound Eco1, averaged from three independent experiments. Error bars represent standard deviations.
(C) Analysis of chromatin from cells synchronized in different cell cycle phases by α factor arrest/release reveal a cell cycle-dependent chromatin association of PCNA and Eco1 and strong binding in S phase. FACS analysis and the levels of the mitotic cyclin Clb2 are used to indicate the cell cycle phase. 10% of the input, 100% of the pellet, and 10% of the supernatant fractions were loaded. The quantifications show the amounts of 3mycEco1 and 3myceco133–281 on chromatin. Bars represent the percentage of chromatin bound Eco1.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 SUMOylation of PCNA Is Detrimental for Eco1 Function
(A) Overexpression of SUMOylation-deficient PCNA mutants (Hoege et al., 2002) leads to a stronger suppression of the temperature sensitivity of eco1-1 and eco1ctf7-203 mutants compared to wt PCNA. In cells deficient in the SUMO ligase Siz1 (responsible for the bulk of PCNA SUMOylation), overexpression of wt PCNA leads to a stronger suppression as well.
(B) Loss of SUMOylation of endogenous PCNA also suppresses the temperature sensitivity of eco1ctf Inactivation of the helicase Srs2, or deletion of its PCNA-SUMO interaction domain (Pfander et al., 2005) (data not shown), does not affect growth of eco1ctf7-203, indicating that the pathway by which PCNA SUMOylation represses eco1 mutants is different from the Srs2-dependent pathway (Pfander et al., 2005), which represses RAD52-dependent recombination at the replication fork.
(C) Loss of PCNA SUMOylation partially rescues the cohesion defects of eco1ctf7-203 at the restrictive temperature (32°C). Sister chromatid cohesion in eco1ctf7-203 and eco1ctf7-203 PCNA-K127,164R cells was quantitatively assayed using yeast strains GFP tagged on ChrIV. Cells were grown either at permissive temperature (26°C) or shifted for 3 hr at 32°C. Each strain was assayed three to five times at both temperatures with at least 50 cells being counted in each experiment. Error bars represent standard deviations.
(D) The PCNA mutant protein, encoded by the pol allele (Amin and Holm, 1996) and introduced in the POL30 locus, shows increased levels of SUMOylation in vivo. Another mutant that is synthetically lethal with eco1ctf7-203, chl1Δ, shows normal levels of PCNA SUMOylation. Cells deficient in PCNA SUMOylation were used as control. The quantification of PCNASUMO levels was done by normalization against the band corresponding to unmodified PCNA and represents an average of three independent experiments. Error bars represent standard deviations.
(E) Ubc9 and Eco1 are rivals for PCNA binding. GST-PCNA was incubated with constant amounts of Eco1 and increasing amounts of Ubc9 (top), or with constant amounts of Ubc9 and increasing amounts of Eco1 (bottom), and subjected to GST pull-downs. As controls, increasing amounts of BSA did not interfere with binding of either Eco1 or Ubc9 to GST-PCNA (Figure S5).
Molecular Cell  , DOI: ( /j.molcel )
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