The Elg1 Replication Factor C-like Complex Functions in PCNA Unloading during DNA Replication  Takashi Kubota, Kohei Nishimura, Masato T. Kanemaki, Anne D. Donaldson  Molecular Cell  Volume 50, Issue 2, Pages 273-280 (April 2013) DOI: 10.1016/j.molcel.2013.02.012 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 50, 273-280DOI: (10.1016/j.molcel.2013.02.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 PCNA and SUMOylated PCNA Accumulate on Chromatin during DNA Replication in the Absence of Elg1 (A) SILAC quantitative proteomic analysis showing chromatin abnormalities in elg1Δ cells undergoing unperturbed S phase (Kubota et al., 2011). Histogram shows log2 ratios of replication proteins identified. Numbers in brackets show number of peptides quantitated for each protein. Strains used are SHY201 and TKY18. (B) PCNA and modified PCNA accumulate abnormally on chromatin in mid-S phase in elg1Δ, shown by western blotting. Cells (SHY201 and TKY18) were released from α factor, samples taken at a mid-S phase time point, and chromatin prepared. Total PCNA (normalized to histone H3) given below was expressed relative to ELG1+. (C) An ELG1-degron strain carrying ELG1-3xmini-AID shows elg1Δ-like MMS sensitivity in the presence of auxin. Serial dilutions (1:5) of cells were grown on YPD + 0.0175% MMS (left) or YPGal + 0.0175% MMS + 0.5 mM auxin (right) at 30°C. (D) Procedure to induce Elg1 degradation. Cells with degron-tagged Elg1 (TKY170) were blocked in G1 phase, and galactose and then auxin added to induce Elg1 degradation before release into S phase. (E) Western blot using anti-AID antibody confirms degradation of Elg1. (F) Chromatin-associated PCNA during replication with or without Elg1. Histone H3 is loading control. PCNA amounts (normalized to histone H3) are indicated below, relative to no degron 0 min sample. (G) Flow cytometry analysis. See also Figure S1. Molecular Cell 2013 50, 273-280DOI: (10.1016/j.molcel.2013.02.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Accumulation of PCNA and SUMO-PCNA on Chromatin in Cells Lacking Elg1 Is Due neither to Delayed Okazaki Fragment Processing nor to DNA Damage (A) Outline. Strains used are TKY166, TKY169, TKY170, and TKY18. (B) Okazaki fragment detection assay. Immature Okazaki fragments were not observed in the absence of Elg1 but were abundant in S phase cells depleted for Cdc9 (DNA ligase I). (C) Flow-cytometric analysis. (D) Western blot to detect Rad53. Phosphorylated Rad53 was not detected in the absence of Elg1. Rad53-P, phosphorylated Rad53; arrowhead, unmodified Rad53. (E) Immunoblot of PCNA in whole-cell extracts. Depletion of either Elg1 or Cdc9 leads to accumulation of SUMOylated PCNA during DNA replication, indicative of increased PCNA association with chromatin. See also Figure S2. Molecular Cell 2013 50, 273-280DOI: (10.1016/j.molcel.2013.02.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Unloading of PCNA Induced by Elg1-RLC In Vivo and In Vitro (A) Procedure for Elg1, Ctf18, Rad24, or Rfc1 overexpression in an elg1Δ background. (B) Chromatin-associated PCNA before (35 min) and after (65 min) GAL induction. Total PCNA on chromatin (normalized to histone H3) is given below, relative to no switch-on. (C) Procedure for switching on Elg1 during or after replication. Cells released in the presence of nocodazole. (D–F) Immunoblot examining SUMOylated PCNA in whole-cell extracts. (D) No expression; (E) Elg1 expression switched on at 35 min; (F) Elg1 expression switched on at 65 min. Lower panels show Elg1-myc expression. (G) SYPRO Ruby-stained gel showing mock IP and IP-purified Elg1-RLC. Asterisk indicates nonspecific background protein. (H) Outline of PCNA unloading assay. (I) Western blot analysis of PCNA on chromatin after treatment with mock IP or Elg1-RLC samples. Total PCNA on chromatin (normalized to histone H3) below, relative to mock IP. See also Figure S3. Molecular Cell 2013 50, 273-280DOI: (10.1016/j.molcel.2013.02.012) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Elg1 Prevents Retention on Chromatin of Both Unmodified and SUMOylated PCNA (A) PCNA in whole-cell extracts (left) and chromatin-enriched fractions (right) prepared from POL30 or pol30 K164K K127R strains that are ELG1 (+) or elg1Δ (−). Cells were released from G1 phase into S phase (200 mM HU) and incubated for 90 min, then whole-cell extracts and chromatin-enriched fractions were prepared. (B) Quantitation of data in (A), normalized to histone H3. (C) Model. Elg1-RLC unloads both unmodified and SUMOylated PCNA (left), so that without Elg1 (right) PCNA is retained on DNA during replication and is consequently SUMOylated. See also Figure S4. Molecular Cell 2013 50, 273-280DOI: (10.1016/j.molcel.2013.02.012) Copyright © 2013 Elsevier Inc. Terms and Conditions