Dynamic Interaction of DNA Damage Checkpoint Protein Rad53 with Chromatin Assembly Factor Asf1  Andrew Emili, David M Schieltz, John R Yates, Leland H Hartwell  Molecular Cell  Volume 7, Issue 1, Pages 13-20 (January 2001) DOI: 10.1016/S1097-2765(01)00150-2

Figure 1 Purification and Analysis of a Rad53–Asf1 Protein Complex (A) Coomassie brilliant blue detection of affinity-purified Rad53 (lane 2) and Skp1 (lane 1) interacting yeast proteins fractionated by SDS–PAGE. The polypeptide bands corresponding to native Asf1 and epitope-tagged Rad53 and Skp1 are highlighted with arrows. Molecular sizes are indicated on the left in kDa. For mass spectrometry, polypeptides copurifying with Rad53 were excised, digested with trypsin, and eluted. Asf1-derived peptides were identified by tandem mass spectrometry and database searching (Grant et al. 1998). (B) Coomassie blue staining of affinity-purified epitope-tagged Rad53 (lane 4), Asf1 (lanes 2 and 3), and control Mre11 (lane 1) protein complexes. Protein identities are given to the right. Variable yields of the slower-mobility overexpressed Asf1 polypeptide have been seen between purifications. Molecular Cell 2001 7, 13-20DOI: (10.1016/S1097-2765(01)00150-2)

Figure 2 Association of Endogenous Rad53 and Asf1 In Vivo (A) Coimmunoprecipitation and Western blot analysis of native yeast gene products. Cell extracts were prepared from strains expressing epitope-tagged Rad53HA, Asf1FLAG, and/or Rad9HA proteins and precipitated with monoclonal antibodies as indicated followed by Western blot analysis. (B) Evaluation of Rad53HA and Asf1FLAG protein levels in a yeast whole-cell extract before (lane 1) and after incubation with anti-MYC (lane 2), anti-HA (lane 3), and anti-FLAG (lane 4) monoclonal antibodies. (C) Immunoprecipitation of Rad53HA from an extract prepared from yeast cells coexpressing Asf1FLAG using either anti-HA (lanes 1 and 2) or anti-FLAG (lanes 3 and 4) antibodies both before (lanes 1 and 3) and after (lanes 2 and 4) exposure to 200 mM hydroxyurea (HU) for 1 hr. Molecular Cell 2001 7, 13-20DOI: (10.1016/S1097-2765(01)00150-2)

Figure 3 Dynamic Interaction of Rad53, Asf1, and Acetylated Histones H3/H4 in Response to DNA Damage and Stalled DNA Replication (A) Coimmunoprecipitation and Western blot analysis of Asf1FLAG and acetylated histone H3 proteins isolated with anti-FLAG antibody from isogenic wild-type and rad53 (mec2-1) mutant cell extracts before (lanes 1 and 4) and after (lanes 2, 3, 5, and 6) exposure to 4-NQO (3 μg/ml) for the indicated time in minutes. The anti-acetylated histone H3 polyclonal antibody used to probe the blot was a generous gift of D. Gottschling. (B) Western blot analysis of acetylated histone H3 complex bound to Asf1FLAG isolated with anti-FLAG antibody from wild-type (sml1Δ), rad53Δ, and mec1Δ mutant cell extracts. (C) Western blot analysis of soluble acetylated histone H3 levels in crude whole-cell lysates made from log phase cultures of isogenic wild-type (sml1Δ) and asf1Δ, rad53Δ, and mec1Δ mutant strains. (D) Coimmunoprecipitation analysis of endogenous Rad53HA, Asf1FLAG, and acetylated histone H3 complexes isolated using either anti-FLAG (lanes 1–3) or anti-HA (lanes 4–6) antibody from cell lysates of log phase cultures before and after exposure to 100 mM hydroxyurea for the indicated time in minutes. (E) Western blot analysis showing modification of endogenous Asf1 in response to stalled DNA replication after exposure of log phase cultures of isogenic wild-type, rad53Δ, and mec1Δ strains to 100 mM HU for 1 hr. Molecular Cell 2001 7, 13-20DOI: (10.1016/S1097-2765(01)00150-2)

Figure 4 DNA Damage Sensitivity of asf1 and rad53 Mutants (A) Growth of 1:3 serial dilutions of wild-type and the indicated mutant yeast strains spotted onto complete media and incubated at 37°C or 30°C in the absence (−) or presence of MMS (0.014%), HU (150 mM), or after UV irradiation (200 J/m2). (B) Cell viability following α factor arrest and synchronous release of isogenic wild-type and asf1Δ, rad53 Δ, and asf1Δ rad53Δ mutant yeast strains into media containing MMS (0.03%). Each time point represents the mean of two independent experiments, and the standard deviation is given for each data point. (C) Plate streakout analysis showing enhanced temperature sensitivity of asf1Δ cdc13 double mutants on rich media relative to wild-type and single mutant strains. Strain identities are provided in the upper left quadrant. Growth was scored after 48 hr incubation at the indicated temperatures. Molecular Cell 2001 7, 13-20DOI: (10.1016/S1097-2765(01)00150-2)

Figure 5 Normal Checkpoint Signaling in asf1Δ Mutants (A) Microscopic analysis showing accumulation of checkpoint (G2/M)-arrested cdc13 and cdc13 asf1Δ mutant cells at 37°C. (B) Western blot analysis of the status of Rad53 and control Rad24 protein in log phase isogenic wild-type and asf1Δ mutant cells before and after exposure to MMS (0.1%). (C) Flow cytometry analysis of the DNA content of asynchronous (“Asyn”) and α factor synchronized strains showing accumulation in S phase of asf1Δ and wild-type cells but not rad53Δ mutants at the indicated times following release into MMS (0.03%). The positions of G1 and G2 peaks are indicated by stippled lines. Molecular Cell 2001 7, 13-20DOI: (10.1016/S1097-2765(01)00150-2)

Figure 6 Rad53 Inhibits Asf1 Chromatin Assembly Activity (A) In vitro chromatin deposition assay wherein Asf1 deposits acetylated histones H3/H4 onto relaxed plasmid DNA. Reactions were as follows: input relaxed closed circular plasmid DNA (lane 2, input), supercoiled plasmid DNA (lane 3), or input closed circular plasmid DNA incubated with either buffer alone (lane 4) affinity-purified Skp1 (lane 5), or affinity-purified Asf1/histone complex (lanes 6 and 7). The products were run out on a nondenaturing agarose gel. The positions of DNA sizing markers, linearized plasmid DNA (Lin), supercoiled plasmid DNA (sc), relaxed input closed circular plasmid DNA (cc), as well as the slower-mobility Asf1-dependent chromatin complexes (pc) are highlighted. (B) Western blot of titrations (1× to 3× amounts) of partially purified Asf1 isolated from wild-type cells (lanes 4–6) or from cells overexpressing Rad53 (lanes 1–3). The positions of Asf1 and Rad53 are indicated. (C) In vitro chromatin deposition assay following incubation of relaxed closed circular plasmid DNA with the indicated relative amounts of either Spk1 (lane 1), Asf1/histone complex (lanes 2–4), or copurified Asf1 and Rad53 proteins (lanes 5–7), respectively. A marked reduction of Asf1-dependent chromatin complexes (pc) can be seen. Molecular Cell 2001 7, 13-20DOI: (10.1016/S1097-2765(01)00150-2)