Volume 45, Issue 5, Pages 696-704 (March 2012) Replisome Stability at Defective DNA Replication Forks Is Independent of S Phase Checkpoint Kinases  Giacomo De Piccoli, Yuki Katou, Takehiko Itoh, Ryuichiro Nakato, Katsuhiko Shirahige, Karim Labib  Molecular Cell  Volume 45, Issue 5, Pages 696-704 (March 2012) DOI: 10.1016/j.molcel.2012.01.007 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Replisome Stability at Defective DNA Replication Forks Is Independent of Mec1 and Rad53 (A) Control (YGDP196), mec1Δ (YGDP198), and rad53Δ (YGDP201) strains were grown at 24°C in YPD medium and synchronized in G1 phase with mating pheromone. Cells were then released for 90 min into fresh medium containing 0.2 M hydroxyurea (HU) before samples were taken and used for immunoprecipitation of TAP-Sld5. The indicated proteins were detected by immunoblotting. (B) The behavior of mec1-100 (YGDP1208) and sld3-A dbf4-4A (YGDP1061) was compared to rad53Δ and mec1Δ in a repeated version of the above experiment. (C) (Ci) Similar experiments with YGDP196 were performed using harsher extract conditions that disrupted replisomes in vitro, unless cells were treated before extraction with a crosslinking agent to preserve in vivo interactions among replisome components. (Cii) The same crosslinking and extract conditions were used to show that replisome material persisted in vivo when control (YGDP196) or mec1Δ (YGDP198) was released into S phase for 90 min in the presence of 0.2 M HU. (D) sld3-7td (YGDP406), mec1Δ sld3-7td (YGDP408), and rad53Δ sld3-7td (YGDP410) were grown at 24°C in YPRaff medium and synchronized in G1 phase, before release for 75 min into YPRaff medium containing 0.2 M hydroxyurea. Expression of GAL-UBR1 was induced at 24°C for 30 min in YPGal medium + 0.2 M HU, before shifting cells to fresh YPGal + 0.2 M medium at 37°C for the indicated times and processing as above. See also Table S1. Molecular Cell 2012 45, 696-704DOI: (10.1016/j.molcel.2012.01.007) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 The Replisome Is Still Associated with Chromosomal DNA When Cells Lacking Checkpoint Kinases Are Subjected to Replication Stress (A) Cells (YGDP101, YGDP102, and YGDP106) were released into S phase in the presence of 0.2 M HU as in Figure 1A above. The extracts were either treated with DNase or left untreated before isolation of replisome material by immunoprecipitation of TAP-Sld5. (B) mec1Δ TAP-SLD5 (YGDP865) and mec1Δ TAP-SLD5 CDC45-5FLAG (YGDP767) were released into S phase for 90 min in the presence 0.2 M HU, and TAP-SLD5 isolated as above. The purified material was released from the IgG resin with TEV protease and then added to extracts of mec1Δ TAP-SLD5 CDC45-18MYC (YGDP796), which were either treated with DNase or left untreated, before immunoprecipitation with anti-FLAG or anti-MYC beads. (Bi) Experimental scheme. (Bii) Material isolated in the first step after immunoprecipitation of TAP-Sld5. (Biii) Material isolated in the final step. See also Table S1. Molecular Cell 2012 45, 696-704DOI: (10.1016/j.molcel.2012.01.007) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Systematic Analysis of Replisome Stability and Progression by ChIP-Seq Control (YGDP415), rad53Δ (YGDP419), and mec1Δ (YGDP417) were grown at 24°C and released into S phase for 60 min in the presence of 0.2 M HU. Following crosslinking with formaldehyde, Mcm4-5FLAG or Pol1-6HA was isolated from sonicated cell extracts and the associated DNA was detected by massive DNA sequencing. The figures show the “enrichment ratio” of the DNA sequence reads for the immunoprecipitates and whole-cell extract samples, calculated as described in the Experimental Procedures, revealing the genomic loci associated with Mcm4 and Pol1 in the three strains. (A) Three early origins on chromosome 6. Green lines indicate the location of tRNAs. (B) Three late/silent origins on chromosome 2. (C) Behavior of replisome components at ARS305 on chromosome 3, one of the earliest origins in the genome. See also Figure S1. Molecular Cell 2012 45, 696-704DOI: (10.1016/j.molcel.2012.01.007) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Mec1-Dependent Phosphorylation of the Psf1 Subunit of the Cdc45-MCM-GINS Helicase at Forks (A) Control (YGDP415), rad53Δ (YGDP419), and mec1Δ (YGDP417) were released from G1 arrest at 24°C into S phase for 30 min (S) or else for 90 min in the presence of 0.2 M HU (HU). After digestion of chromosomal DNA, Mcm4 was isolated from cell extracts by immunoprecipitation. (B and C) Mec1-dependent phosphorylation of Psf1 is independent of the checkpoint kinases Rad53, Chk1, Dun1, and Tel1. (D) The increased fraction of phosphorylated Psf1 in rad53Δ cells is not due to processing of replication forks by the Exo1 nuclease. (E) More of MCM-associated Psf1 is phosphorylated in cells that fire both early and late origins. (F) Enhanced phosphorylation of γ-H2A in rad53Δ but not sld3-A dbf4-4A. Asterisks mark a background band that is recognized in cell extracts by the Psf1 antibody, and arrows denote the hyperphosphorylated form of Psf1. Molecular Cell 2012 45, 696-704DOI: (10.1016/j.molcel.2012.01.007) Copyright © 2012 Elsevier Inc. Terms and Conditions