BAH domain structure and mutations. (A) Computer generated diagrams of the BAH domains in ScOrc1 (aa 53–176), ScRsc2 (aa 412–514) and ScSir3 (aa 53–176) were obtained using CPHmodels 2.0 Server and DeepView Swiss-Pdb Viewer. Green and blue ribbons indicate α-helix and β-sheet structures, respectively. Van der Waals surfaces of a highly conserved glutamic acid residue are indicated. (B) BAH domains were identified using ELM-‘functional sites in proteins' program. ScOrc1 and HsOrc1 BAH-domains consist of 10 β-sheets and three α-helices. Numbers refer to the amino-acid sequence. The conserved glutamic acid is in β-sheet no. 7 in ScOrc1 (Zhang et al, 2002) and no. 5 in chicken polybromodomain protein (Oliver et al, 2005). (C) HsOrc1 landmarks include BAH and AAA+ domains. A nuclear localization signal and a HP1 binding site have been reported (Lidonnici et al, 2004). FH-Orc1(wt)=aa 1–861. FH-Orc1(ΔBAH)=(Δ(1–169)). FH-Orc1(ΔH)=(Δ(98–107)), and FH-Orc1(E111K)=E → K at position 111), N315=aa 1–315. Abbreviations are Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Xl, Xenopus laevis; Mm, Mus musculus; Cg, Cricetulus griseus; Rn, Rattus norvegicus; Cf, Canis familiaris; Pt, Pan troglodytes; and Hs, Homo sapiens.

The HsOrc1 BAH domain facilitated association of Orc1 with chromosomes in vivo. Exponentially proliferating cells expressing either FH-Orc1 wt, ΔBAH, E111K or ΔH were fixed with formalin, permeabilized with SDS buffer, and stained with propidium iodide to detect nuclear DNA (red) and with anti-HA to detect recombinant protein (green). Images were captured in a confocal microscope and then merged to reveal sites where DNA and FH-Orc1 protein were coincident (yellow). Interphase nuclei containing FH-Orc1 were in G1-phase (G1), because they lacked PCNA (see Figure 2C). Cells with a puddle of condensed chromatin were considered prophase (P). Cells with condensed chromosomes aligned in a single plate were considered metaphase (M). Cells with two metaphase plates were considered anaphase (A). Two adjacent cells, each with condensed chromosomes in a single plate of condensed chromosomes, were considered telophase (T). Similar results were obtained with cells fixed in formalin and permeabilized with Triton X-100, and with cells fixed in methanol only.

HsOrc1 facilitated association of Orc2 with chromosomes in vivo. (A) Cells expressing FH-Orc1(wt) were lysed, extracted with increasing amounts of salt, and then subjected to immuno-blotting using anti-Orc2 antibody, as in Figure 4. (B) Exponentially proliferating 293EBNA1 cells (control) or 293EBNA1 cells expressing either FH-Orc1 wt or E111K were stained with DAPI to detect nuclear DNA (blue), anti-HA to detect FH-Orc1 (green), and anti-HsOrc2 (red), as in Figure 3.

BAH domain in HsOrc1 facilitated oriP-dependent plasmid DNA replication in human cells. (A) Plasmid maintenance assays: 293EBNA1 cells constitutively expressing either FH-Orc1 wt, ΔBAH, E111K or ΔH were co-transfected with the oriP-dependent plasmid p818 (Figure 6A) and pEGFP, a plasmid that lacks oriP. Cells were selected for their resistance to hygromycin B. Cells transformed with pOP, the parental expression vector, were used as a control. Results were normalized to the fraction of eGFP expressing cells. (B) Transient plasmid DNA replication assays: 293EBNA1 cells constitutively expressing either FH-Orc1 wt, ΔBAH, E111K or ΔH were co-transfected with p818 to determine the effect of the ectopic protein on oriP-dependent DNA replication, and with pEGFP to measure transfection efficiency. Plasmid DNA was isolated 4 days post-transfection, and then digested with BamH1 to convert it into linear monomers and with DpnI to degrade unreplicated DNA. DNA was detected by blotting-hybridization using appropriate ³²P-DNA probes and quantified using a phosphorimager. Each lane contains DNA from an equivalent number of transfected cells. ‘STD' is purified p818. Transfections were carried out in duplicate. Error bars indicate s.e.m. for four independent experiments.

‘ORC cycle' in mammalian cells (reviewed in Depamphilis et al, 2006). ORC is bound to chromatin during G1-phase of the cell cycle where it is part of a prereplication complex. When S-phase begins, human Orc1 is selectively degraded by the 26S proteasome via a ubiquitin-dependent mechanism, and then reappears during the M to G1-phase transition. During metaphase, Orc1 is hyperphosphorylated, an event that prevents ORC assembly. As cells exit metaphase, Orc1 is dephosphorylated and, together with other ORC subunits, binds to chromatin, an event that is facilitated by the Orc1 BAH domain.

Effects of BAH domain mutations in HsOrc1 on FH-Orc1 expression, degradation, and ORC assembly. (A) Relative levels of the indicated FH-Orc1 protein were determined by subjecting whole-cell lysates to immuno-blotting using anti-HA antibody to detect recombinant proteins and anti-HsOrc2 to detect endogenous Orc2. Cells transformed with pOP served as a control. (B) Cells cultured in 25 μM MG132 for 4 h before lysis were subjected to immuno-blotting. (C) Asynchronously proliferating cells were stained with anti-HA antibody to detect FH-Orc1(wt) (red), anti-PCNA antibody to detect PCNA (green), and DAPI to visualize nuclear DNA (Blue). (D) Cells expressing either FH-Orc1 wt or ΔBAH were fractionated by centrifugal elutriation, and individual fractions were analyzed by FACS. Nuclei from cells lysed in HB were subjected to immunoblotting. (E) FH-Orc1 wt and ΔBAH were affinity purified from NEB350 extracts of 293EBNA1 cells that constitutively expressed the indicated protein. FH-Orc1 was bound to anti-FLAG M2 agarose beads, eluted with FLAG peptide, fractionated by SDS-gel electrophoresis and stained with silver. The positions of FH-Orc1 (100 kDa), Orc3 (82 kDa), Orc2 (66 kDa), Orc4 (50.4 kDa), Orc5 (50.3 kDa) and Orc6 (28 kDa), and Mark 12 protein standards (Invitrogen) are indicated. Experimental details are provided in ‘Supplementary Materials and methods'.

The HsOrc1 BAH domain stabilized the association between Orc1 and nuclear components in cell lysates. (A) The level of FH-Orc1 in 293EBNA1 cells that constitutively expressed this protein was compared directly with the level of endogenous Orc1 in 293EBNA1 cells that had been transformed with the expression vector pOP. Identical extracts from the same number of cells were fractionated on the same gel before subjecting it to immunoblotting with anti-HsOrc1 antibody. Extract from FH-Orc1 cells was serially diluted before fractionation. (B) Cells expressing the indicated protein were fractionated into cytosol and nuclei as previously described (Mendez and Stillman, 2000), with modifications (see Supplementary Materials and methods). Cells were lysed in HB and then sequentially extracted with NEB containing either 170, 350 or 500 mM NaCl. The soluble fractions from each extraction as well as the final nuclear pellet were then subjected to Western immunoblotting either with anti-HA antibody to detect FH-Orc1, or with anti-HsMcm3 antibody. HsMcm3 was assayed in the FH-Orc1(wt) cell line. Proteins were identified on the basis of their size and antibody crossreactivity.

The HsOrc1 BAH domain facilitated binding of Orc1 to oriP DNA. Cells constitutively expressing either FH-Orc1 wt, ΔBAH, E111K or ΔH and maintaining p818 as an episome were subjected to chromatin immunoprecipitation (ChIP) assays using anti-HA antibody, as previously described (Chaudhuri et al, 2001; Schepers et al, 2001), with modifications (see Supplementary Materials and methods). (A) The relative locations of PCR probes E15′ and SC5 in plasmid p818 are indicated along with the EBNA1 and hygromycin phosphotransferase (hpt) genes (transcription indicated by arrow), and the oriP FR and dyad symmetry (DS) elements. SC5 is about 4 kb from E15′. (B) FH-Orc1 and Orc2 recovered in ChIP assays were detected by immunoblotting with a mixture of anti-HsOrc1 and anti-HsOrc2 antibodies. (C) Relative amounts of p818 DNA containing probe E15′ (gray) and SC5 (cross-hatched) sequences recovered from cells expressing FH-Orc1(wt) were determined by real-time quantitative PCR and expressed as a fraction of p818 episomal DNA. (D) The experiment in (C) was carried out with cells expressing either FH-Orc1 wt, ΔBAH, E111K or ΔH. Error bars indicate s.e.m. for four independent experiments.

The N-terminal fragment of HsOrc1 containing the BAH domain inhibited oriP-dependent plasmid DNA replication. 293EBNA1 cells were transfected in triplicate with p818 (Figure 6A), either in the presence or absence of pF-N315, a plasmid expressing FLAG-tagged N315, the N-terminal 315 amino acid portion of FH-HsOrc1 (Figure 1C). Four days post-transfection, whole-cell extracts were subjected to immunoblotting using anti-FLAG and anti-CcnA2 antibodies (A), stained with anti-FLAG antibody (green) to determine whether or not N315 localized to the nucleus (B), and extracted first with HB and then with NEB containing the indicated mM amount of NaCl (C). (D) Transient plasmid DNA replication assays were carried out as in figure 7B. (E) Total p818 and pEGFP DNA (−DpnI) in either the presence or absence of pF-N315 was quantified using a phophorimager. (F) Replicated p818 (black bars) and pEGFP (gray bars) DNA was quantified as the amount of DNA resistant to DpnI digestion relative to the total amount detected in the absence of pF-N315.