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. 2008 Feb 5;105(5):1692-7.
doi: 10.1073/pnas.0707260105. Epub 2008 Jan 30.

Interaction Between HMGA1a and the Origin Recognition Complex Creates Site-Specific Replication Origins

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Free PMC article

Interaction Between HMGA1a and the Origin Recognition Complex Creates Site-Specific Replication Origins

Andreas W Thomae et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G(1) and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HMGA1a supports plasmid replication and interacts with ORC. (A) Design of the transacting factors HMGA1a:EBNA1-DBD and EBNA1. HMGA1a (green) was fused to the DNA binding and dimerization domain (EBNA1-DBD, gray) and nuclear localization signal (black) of EBNA1. AT hooks are depicted in blue, HMGA1a's acidic domain in orange. The N-terminus of EBNA1 contains two linking domains [light red and green (LR1 and LR2)]. Each LR comprises a Gly-Arg-repeat. HEK293 cells (293) and derivatives, expressing either EBNA1 (EBNA1) or HMGA1a fused to EBNA1-DBD (HMGA1a:EBNA1-DBD), were transfected with the oriP plasmid shown. After selection, low-molecular-weight DNA was isolated and digested with DpnI. E. coli DH10B cells were electroporated with 500 ng of DNA, and ampicillin-resistant colonies of three independent experiments were counted. Mean and standard deviations are provided. (B) HMGA1a interacts with ORC subunits. For affinity purification experiments, HMGA1a:EBNA1-DBD with a C-terminal Strep-tagII was stably introduced in HEK293 cells. Pull-down experiments with nuclear protein of 2 × 107 cells indicate an interaction of HMGA1a:EBNA1-DBD and different ORC subunits (lane 6). Western blots of nuclear extracts of different cell numbers are shown in lanes 1–3. An Orc2-specific antibody was used to coprecipitate HMGA1a:EBNA1-DBD from 2 × 107 cells in lanes 8–10, which was detected with an EBNA1-specific antibody. Protein G Sepharose beads were eluted with 5% N-Lauroylsarcosine, which preferentially released HMGA1:EBNA1-DBD but not Orc2 as shown in lane 8. Remaining Orc2 was eluted with Laemmli buffer (IP; lane 9). The unbound fraction of the protein lysate is shown in lane 10 (unbd). An isotype antibody did not precipitate HMGA1a:EBNA1 (lane 7). (C) Endogenous Orc6 and HMGA1a coprecipitate (Left) and interact directly in the absence of DNA (Right). Nuclear extracts of 2 × 107 HeLa cells were precipitated with an HMGA1a-specific rabbit antibody or an isotype control. The former coprecipitated Orc6 (Left). Nuclear extracts of 2 × 104 and 2 × 105 cells are shown for comparison. In pull-down experiments, recombinant His-tagged Orc6 (purified from Baculovirus-infected insect cells) was coprecipitated with bacterially expressed and highly purified Strep-tagged HMGA1a protein (10 μg each) immobilized to Strep-Tactin (IBA) (Right). Ten and one nanograms of recombinant Orc6 protein samples are shown for comparison.
Fig. 2.
Fig. 2.
BiFC analysis and colocalization of HMGA1a and Orc2. Fluorescence images of HepG2 cells expressing the indicated proteins were acquired 24 h after DNA transfection. (A) Analysis of interaction of HMGA1a with Orc1 and Orc6. HMGA1a fused to the N-terminal domain of YFP was cotransfected with Orc1 (I) and Orc6 (II) linked to the C-terminal fragment of YFP. (III) An HMGA1a variant with mutated AT-hooks (R3xG) was cotransfected with the Orc6-BiFC construct. To calculate BiFC efficiencies, cells were cotransfected with BiFC vectors and an mRFP-expression vector. For example, for Orc1, 32.5% of all cotransfected cells (n = 151) showed BiFC and 70% of BiFC-positive cells showed the depicted pattern. For intensity profiles, see SI Figs. 8 and 9. (B) Colocalization of Orc2 and HMGA1a-eGFP was visualized in fixed HepG2 cells by immunofluorescence using an Orc2-specific antibody. HMGA1a-eGFP and Orc2 colocalize especially in perinucleolar regions (arrows). (C) HMGA1a and Orc6 BiFC experiments in the presence of 5 μg/ml Hoechst 33342 incubated for 1 (a) and 10 min (b). (Scale bars: 10 μm.) (c) Signal complexes of HMGA1a/Orc6 BiFC- and HP1α-mRFP partially overlap. Orc6 and HMGA1a-BiFC plasmids were cotransfected with an HP1α-mRFP expression plasmid (see also SI Fig. 9D). To increase contrast, the yellow BiFC signal was changed to green. (D) Orc4/Orc6 and Orc5/Orc6 BIFC-signals altered after overexpression of HMGA1a-mRFP. Orc4/Orc6 (a) and Orc5/Orc6 were cotransfected into HepG2-cells. Cotransfection of the indicated BiFC-plasmids (a′ and b′) and HMGA1a-mRFP (a″ and b″) resulted in a significant relocalization of ORC (a‴ and b‴). In A–C, Left shows interference contrast figures of the transfected cells. (Scale bars: 10 μm.)
Fig. 3.
Fig. 3.
HMGA1a mediates conditional DNA replication. (A) scTetR::HMGA1a consists of a single-chain (sc) dimer of the DNA-binding domain of the tetracycline repressor (TetR; red) fused via an artificial linker [(SG4)5, yellow] to the entire HMGA1a coding sequence. HEK293 cells (lanes 1 and 3) and a derivative expressing both EBNA1 (lane 2) and scTetR:HMGA1a (lane 4) were analyzed with EBNA1- and TetR-specific antibodies in Western blots. HEK293 cells expressing scTetR:HMGA1a (lane 6) and scTetR (lane 7) were analyzed with a TetR-specific antibody. (B) OriP has a bipartite structure: FR is an array of 20 high-affinity-EBNA1-binding sites (black circles); DS encompasses two pairs of EBNA1-binding sites (black circles). Four tet-operator sites (tetO4, red circles) replace DS in FRwttetO4. FRwttetO32 contains eight tetO-clusters. (C) Reporter plasmids were transfected into EBNA1+/scTetR+- (Left) or EBNA1+/scTetR:HMGA1a+-HEK293 cells (Right) and selected for 2–3 weeks (sel.) Low-molecular-weight DNA was prepared and digested with DpnI and HindIII. The radioactive probe used in the Southern blot hybridizations recognized a DNA fragment of 3.5 kbp (black arrowhead). A background signal appears at 2.5 kbp (open arrowhead). Addition of doxycyclin (+; 2 μg/ml) without hygromycin selection caused a modest reduction in copy number of the FRwttetO plasmids at the indicated time points (7 and 13 days). The drug did not affect the copy number of the oriP control plasmid, as indicated by the relative signal intensities in percent.
Fig. 4.
Fig. 4.
Multiple tetO sites increase the copy number of scTetR:HMGA1a-dependent plasmids and recruit ORC to the origin of DNA replication in a dose-dependent manner. (A) Different reporter plasmids with one (FRwttetO4), two (FRwttetO8), four (FRwttetO16), and eight (FRwttetO32) arrays of tetO4-motifs. (B) EBNA1+/scTetR:HMGA1a+-HEK293 cells were transfected with the indicated plasmid and replication was assessed as described in Fig. 3. (C) ChIP analysis indicates site-specific ORC binding. For each experiment, 500-μg chromatin of HEK293 cells stably transfected with the indicated plasmids was used. The location of the primer pairs is designated: DS is in close proximity to the tetO array, control primer pairs 1 and 2 are located 1.4 kbp downstream and 2.0 kbp upstream of the tetO sites, respectively. The heights of the columns indicate the relative enrichment (mean values and standard deviation of three experiments) on a logarithmic scale expressed as the difference between PCR values (Cp) obtained with the Orc3-specific antibody vs. controls obtained with preimmune serum (IgG). (D) High local concentration of HMGA1a promote site-specific initiation. A pEPI-based plasmid (pEPI-tetO20; Left) encompassing an array of 20 tetO sites. Nascent-strand analysis was performed in HCT116 cells and a derivative expressing scTetR:HMGA1a. Nascent DNA was purified from parental HCT116 cells (green bars) and scTetR:HMGA1a+ HCT116 (red bars) stably transfected with pEPI-tetO20. DNA was quantified by real-time PCR 2 weeks after transfection. The histogram shows the relative ratio of three different primer sets. The primer pair polyA was arbitrarily set to one.

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