PMC

The current view on the different associations of Hat1p within the cell. In addition to the cytoplasmic Hat1p/Hat2p complex, the Hat1p/Hat2p-Hif1p chromatin assembly complex is recruited to chromatin during S-phase. A minor fraction of Hat1p/Hat2p is constitutively associated with ORC and presumably chromatin-bound. Additional H3-specific acetylation and additional substrates for Hat1p are possible.

ORC-associated histone acetylation. (A) Incorporation of ¹⁴C-AcCoA into core histones from chicken erythrocytes by ORC-associated histone acetyltransferase. The upper panel shows PhosphorImage of ¹⁴C-labeled histones and the lower panel shows Coomassie stained gel. Protein introduced in the assay was 10, 5, 2, and 1.25 ml of the TAP-purified and concentrated product (approximately 100 ml). (B) Quantification of the results in panel A. Disintegrations/decays per min are shown on the Y-axis. (C) Equal volumes of TAP-purified proteins used for the HAT assay from ORC5-TAP (BSY700), ORC5-TAP hat1Δ (BSY701), and untagged control strain (JRY2334) are loaded on a silver stain gel.

A genetic interaction between orc-ts alleles and mutations in the B-type histone acetyltransferase. (A) Analysis of tetrads from a cross of orc5-1 (JRY4250) with hat1Δ (BSY539) and hat2Δ (BSY540). Spores were incubated for 3 days at 31°C, which is the semi-permissive temperature for orc5-1. Double mutants are marked with circles. (B) Plating assay of orc5-1 hat1Δ (BSY538), orc5-1 hat2Δ (BSY602), and orc5-1 hat1Δhat2Δ (BSY603, BSY604) with control strains at indicated times and temperatures. Dilutions were 1:10, starting from late log-phase cultures on YPD. (C) Plating assays for orc2-1 mutant combinations with hat1Δ (BSY569) and hat2Δ (BSY572). (D) Plating assays as in B for orc1-161 mutant combinations with hat1Δ (BSY589-BSY591). (E) Growth comparison of orc5-1 hif1Δ (BSY595) with orc5-1 hat1Δ and orc5-1 hat2Δ at 30°C. For a detailed list of strains, see Additional file .

The constitutive association of Hat1p/Hat2p with ORC. (A) Co-immunoprecipitation (IP) of Hat1p with ORC from log-phase cells. Strains were: untagged control (JRY2334), Hat1–13myc (BSY676), and Orc5–13myc (BSY677). IPs were performed with monoclonal a-Orc3p and a-GFP antibodies. One twentieth of the extracts were loaded as whole cell extract (WCE). Immunoprecipitated Hat1–13myc was detected with mouse α-myc as a primary antibody (9E10). Note that equal amounts and concentrations of extracts were used for all IPs. (B) ORC binds Hat1p/Hat2p but not Hif1p. Strains were: untagged control, Hat1–13myc, Hat2–13myc (BSY691), and Hif1–13myc (BSY692). One twentieth of the extracts were loaded as WCE control. IPs were performed with monoclonal α-Orc3p and α-GFP control antibodies. (C) IP of Hat1p-13myc with α-Orc3p antibody in synchronized cells. Log-phase cells were either arrested in 3 μg/ml α-factor, 200 mM hydroxyurea, or 15 μg/ml nocodazole. Cells were also released from α-factor arrest and samples were taken at 20, 30, and 40 min after release (lanes 4–6). Lanes 7–12 show WCE controls. (D) FACS control of samples in panel C. Time after release is indicated (Rel).

Analysis of DNA replication phenotypes. (A) Plasmid loss rates were measured in orc5-1 hat1, orc5-1, hat1, and wild-type control strains by growing the cells for approximately 10 generations. Strains were JRY2334 (W303 wild-type control), BSY528 (hat1), BSY535 (orc5-1), BSY538 (orc5-1 hat1). These strains were transformed with plasmids pDK243, pDK368-1, pDK368-2, pDK368-7, containing 1, 2, 3 or 8 ARS sequences, respectively. Y-axis shows plasmid loss rates (loss frequency/generation). (B,C) Structural analysis of replicative intermediates by two-dimensional gel electrophoresis. Cultures of hat1 (BSY528) and wild-type (JRY2334) strains were released from G1 arrest for 15 and 30 min (30°C). The same blot was first hybridized with an ARS305 specific probe (B), then stripped and rehybridized with an ARS1 specific probe (C). The 15 min wild-type sample is overloaded as judged by the amount of monomers. (D) Scheme for replicative intermediates observed in two-dimensional gel electrophoresis.

Hat1-TAP is recruited to early and late origins at the time of firing. Samples were taken at indicated times and processed for chromatin immunoprecipitation and input controls. DNA was either labeled with ³²P (panels A and C) or visualized by ethidium bromide (panels B and D). (A) Binding of Hat1-TAP and Cdc45-TAP to ARS305 and to R11 control sequence at the time of origin firing and later in S-phase. Strains were HAT1-TAP (BSY679) and CDC45-TAP (BSY680). One representative experiment with input and immunoprecipitate and the percentage of precipitated DNA is shown. (B) Recruitment of Hat1p to ARS1 is coincident with Cdc45p and dependent on functional ORC. Strains used for chromatin immunoprecipitation are HAT1-TAP, CDC45-TAP, and HAT1-TAP orc2-1 (BSY699). Strains were held in a-factor at 36°C to inactivate orc2-1 and then released at 23°C. (C) Recruitment of Hat1p (Hat1-TAP) to ARS1412 late replication origin and comparison with R11 sequence. (D) Recruitment of Hat1p to ARS1 is affected by the cdc7-1 allele. Strains used for chromatin immunoprecipitation are HAT1-TAP, CDC45-TAP, and HAT1-TAP cdc7-1 (BSY734).

Growth defect of orc5-1 ts in combination with histone tail mutants. In a plasmid shuffle assay, orc5-1 strain (AP121) and orc5-1 hat1 strain (AP123) were transformed with TRP-marked plasmids that contain histones H3 and H4 with lysine to arginine substitutions at different sites in their N-terminal tails. (A) Arginine substitution in H4 lysine 5, 12 shows reduced viability in combination with orc5-1 on YPD at the temperature indicated (31°C). Cells were grown for 3 days. (B) The replication defect in orc5-1 leads to efficient loss of the ADE2 marked plasmid with the covering H3/H4 wild-type genes when selection is performed on -TRP medium. When the combination of orc5-1 with histone mutants is nonviable, cells are less likely to lose the covering plasmid. Colonies were grown at 23°C. (C) Direct assays of transformation and loss of wild-type histone plasmid in different H3 mutant backgrounds. H3 histone mutants contain arginines substituted for lysines. (D) Plasmid loss assays with strains (AP182, AP183) containing wild-type histone H3 (pmp3), K9, 14, 18, 23, 27R substituted histone H3 (pmp8), and K14, 23R substituted histone H3 (pmp83). Loss rates were measured for ARSH4 (pRS316) and ARS120 (YCp120). Averages and standard deviations of three independent experiments are shown.

Physical interaction of ORC with the Hat1p/Hat2p complex. (A) The B-type HAT complex was purified from a strain containing a TAP-tagged Hat1p subunit, and ORC was purified from strains carrying either a TAP-tagged version of Orc1p, Orc2p, Orc4p, Orc5p, or Orc6p. For comparison, lane 2 shows marker proteins (MWs are 45.0, 66.2, and 97.4 kDa). The positions of the respective subunits of the ORC and Hat1p complex are indicated. Hat1-CBP: Hat1p with calmodulin binding protein (CBP) after TEV digestion of the TAP-tag. (B) Summary of proteins identified in purifications of TAP-tagged baits. Components of ORC and Hat1p complexes that were detected at least once with either MALDI-TOF or liquid chromatography-mass spectrometry (LC-MS) with high confidence (>90%) are indicated (M/L). (C) Architecture of the Hat1p complexes by differential tagging and subunit deletions. Strains were HIF1-TAP (BSY675), HIF1-TAP hat1Δ (BSY681), HAT1-TAP (BSY679), HAT1-TAP hif1Δ (BSY720), HAT1-TAP hat2Δ(BSY682). Lane 1 shows marker proteins (45.0, 66.2, and 97.4 kDa). (D) Western blot from two series of TAP purifications (Figure 1C for left panel, Additional file for right panel), probed with a-Hat1p, a-Orc2p, a-Orc3p, and a-Orc5p antibodies. (E) In vitro histone acetyltransferase activities of Hat1p complexes. Concentrated eluate (10-fold) from indicated TAP-tag purifications from Figure 1C was used for HAT-assays with ¹⁴C acetyl-CoA and chicken erythrocyte histones. The upper panel shows ¹⁴C incorporation into histones shown in the lower panel by Coomassie strain. (F) In vivo association of acetylated histone H4 with Hat1p sub-complexes from Figure 1C). Eluates from TAP-tag purifications were analyzed for Lys12 acetylated histone H4 by Western blot (a-Acetyl H4 Lys12).

The hat1Δ mutation reduces viability and enhances the cell-cycle defect of orc-ts cells. (A) Percentage of cells that form viable microcolonies when asynchronous cultures of orc5-1 hat1 (BSY538), orc5-1 (BSY535), hat1 (BSY528) and wild-type strain (JRY2334) were shifted to the nonpermissive temperature for 0–5 h. Viability was measured as the fraction of microcolonies that formed after the incubation at 36°C within 1–2 days at permissive temperature. (B) Percentage of orc5-1 hat1, orc5-1, hat1, and wild-type cells that form viable microcolonies when synchronized cultures were shifted to the nonpermissive temperature. Cells were arrested in G1 (α-factor) or in S-phase (hydroxyurea) at 23°C and then maintained at restrictive temperature (36°C) for 0 to 3 h in G1 phase, in S-phase or from G1 to S-phase arrest. Averages of two independent experiments are shown. (C) FACS analysis of wild-type, hat1Δ (BSY539) orc2-1 (BSY568) and orc2-1 hat1Δ (BSY569) cells at semi-permissive temperature (26°C). Cells were arrested in α-factor (5 mg/ml) and release was performed at 26°C for 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, and 190 min. (D) Cell-cycle progression of orc5-1 hat1, orc5-1 and wild-type strain at restrictive temperature for orc5-1. G1-arrested cells were held at 36°C (restrictive temperature) for 1 h and then released into fresh (36°C) medium. Samples for FACS were taken at times indicated. For a detailed list of strains, see Additional file .