Figure 1.

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 1 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).