Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation

Abstract

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G(1)-to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G(1), and with geminin in post-G(1) cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G(1) cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.

Fig 1

ORCA is required for entry into the cell cycle. (Aa) Scheme of the experiment in WI38 cells. Cells were serum starved for 5 days. ORCA or control siRNA was transfected three times at intervals of 24 h starting day 3. On day 6, the cells were released from arrest for fluorescence-activated cell sorter (FACS) or immunoblot analysis. (Ab) Immunoblot showing efficient knockdown of ORCA. (Ac) FACS analysis at 12 h and 24 h postrelease in control and ORCA siRNA-treated cells. Note the efficient release of cells into the cell cycle in the 24-h FACS profile for control cells but a G₁ arrest in ORCA-depleted cells. (Ad) Chromatin fractionation in control and ORCA siRNA-treated cells and immunoblotting with Orc2, MCM3, and geminin. SRSF1 and MEK2 are shown as loading controls for chromatin (P) and cytosolic (S) fractions, respectively. (B) Relative levels of ORCA, Orc1, Orc2, Cdt1, and geminin in asynchronously growing human U2OS cells. GST-tagged ORCA and His-tagged Orc1, Orc2, Cdt1, and geminin were loaded as indicated (ng) for quantitation. (C) Relative levels of ORCA, Orc1, Orc2, Cdt1, and geminin in the G₁ phase of U2OS cells. Note that ORCA, Orc1, and Cdt1 levels are high during G₁, whereas geminin levels are negligible in G₁. Asyn, asynchronous. (D) Relative abundances of ORCA, Orc1, Orc2, Cdt1, and geminin on chromatin (P) during G₁ in human U2OS cells. The asterisks indicate cross-reacting bands. The arrowheads indicate endogenous ORCA (ORCA immunoblot) and endogenous Cdt1 (Cdt1 immunoblot).

Fig 2

Stoichiometry of ORCA bound to ORC, Cdt1, and geminin using SiMPull analyses. (Aa) Schematic representation of the SiMPull assay. (Ab and Ac) Representative single-molecule fluorescence time trajectories of YFP-geminin molecules that exhibit one-step (Ab) and two-step (Ac) photobleaching. (B) ORCA-Orc1 pulldown. (Ba and Bb) Schematic (Ba) and TIRF (Bb) images of YFP molecules pulled down from U2OS cell lysates expressing T7-ORCA and YFP-Orc1 using biotinylated T7 antibody. The lysate expressing only YFP-Orc1 served as the control. (Bc) Average numbers of YFP fluorescent molecules per imaging area (2,500 μm²). The error bars indicate standard deviations of the mean values from 20 imaging areas. (Bd) Photobleaching step distribution for YFP-Orc1 bound to T7-ORCA. (Be) Fluorescence intensity distribution of YFP molecules exhibiting 1 and 2 photobleaching steps. Nearly 15% of the molecules could not be unambiguously scored and were discarded; the discarded molecules showed no enrichment of intensity. (Ca to Ce) ORCA-Cdt1 pulldown. Shown are YFP molecules pulled down from U2OS cell lysates expressing T7-ORCA and YFP-Cdt1 using biotinylated T7 antibody. The same lysate incubated with the biotinylated Flag antibody served as the control. (Da to De) ORCA-geminin pulldown. YFP molecules pulled down from U2OS cell lysates expressing T7-ORCA and YFP-geminin using biotinylated anti-rabbit IgG and ORCA antibody were analyzed. The same lysate incubated with the biotinylated anti-rabbit IgG and rabbit IgG served as the control. The fluorescence intensity of molecules bleaching in two steps was nearly twice that of molecules bleaching in a single step.

Fig 3

ORCA association with ORC, Cdt1, and geminin is cell cycle regulated. (A) Association of ORCA with ORC, Cdt1, and geminin in human cells. Shown are HeLa nuclear extract fractionated over a Superdex 200 gel filtration column and fractions analyzed for ORCA, Orc2, Cdt1, and geminin by immunoblotting. Molecular mass markers are labeled above the blot. (Ba) ORCA immunoprecipitation during different stages of the cell cycle. ORCA associates with Orc2 throughout the cell cycle. ORCA associates with Orc1 during G₁ but not efficiently with the phosphorylated Orc1 during mitosis. ORCA associates with Cdt1 during G₁, as well as robustly with phosphorylated Cdt1 during mitosis. ORCA associates strongly with geminin from G₁/S to mitosis. (Bb) Immunoblot analysis of whole-cell extract from nocodazole-arrested mitotic extracts treated with phosphatase and untreated. Note that the phosphorylated form of Cdt1 and geminin collapses on phosphatase treatment. (C) Glycerol gradient sedimentation analysis of ORCA complex on material (asynchronous sample) immunoprecipitated using ORCA antibodies. The corresponding molecular mass markers are labeled. Note that ORCA and Orc2 cosediment in fractions 11 to 15, ORCA and Cdt1 cosediment in fractions 11 to 14, and ORCA and geminin cosediment in fractions 8 and 13 to 15. Fractions 13 and 14 contain ORCA, Orc2, Cdt1, and geminin, suggesting the existence of a quaternary complex, as well. (D and E) Glycerol gradient sedimentation analysis of ORCA-containing complex from G₁ extracts (D) and mitotic extracts (E). Note the cosedimentation of ORCA-Orc2-Cdt1 (fraction 11) in G₁ extracts and ORCA-Orc2-Cdt1-geminin (fractions 9 and 10) in mitotic extracts. (F) Gel filtration analysis of ORCA-containing complex during mitosis. (G) Sequential immunoprecipitation of ORCA, followed by Cdt1 from mitotic extracts. Immunoblot analysis of geminin corroborated the presence of ORCA-Orc2-Cdt1-geminin complex in mitotic extracts.

Fig 4

ORCA associates with Orc2 directly and utilizes WD to associate with ORC, Cdt1, and geminin. (Aa to Af) Binary infections using ORCA baculovirus in combination with Orc1 (Aa), Orc2 (Ab), Orc3 (Ac), Orc4 (Ad), Orc5 (Ae), and Orc6 (Af) in insect cells, followed by immunoprecipitation using ORCA antibody from whole-cell extracts. Note the prominent interaction between Orc2 and ORCA. (B) Direct interaction between ORCA and Orc2. His-ORCA and His-Orc2 were purified on Talon columns, and the two proteins were incubated; then, immunoprecipitation was carried out using Orc2 antibody. (C and D) Reconstitution of the ORCA-ORC complex. Coinfection by baculovirus carrying ORCA plus Orc1, Orc2, Orc3, Orc4, or Orc5 in insect cells and immunoprecipitation of whole-cell extracts with Orc2 (C) or ORCA (D) antibody. (E and F) Binary infection using ORCA baculovirus in combination with Cdt1 and geminin in insect cells, followed by immunoprecipitation using ORCA antibody from nuclear extracts. (G) Schematic representation of various truncation mutants containing a T7 epitope tag at the N terminus of ORCA. (H) IP in U2OS cells expressing various T7-ORCA mutants and Flag-Cdt1 using T7 antibody and analysis of Cdt1 by immunoblotting. (I) IP in U2OS cells expressing various T7-ORCA mutants and YFP-geminin using T7 antibody and analysis of geminin by GFP immunoblotting. Note that only ORCA constructs possessing the WD40 domain efficiently interact with Orc2 (47), Cdt1, and geminin (T7-ORCA.128-647, T7-ORCA.270-647, and T7-ORCA.1-647). The asterisks indicate cross-reacting bands.

Fig 5

ORCA requires Orc2 for its stability. (A) Schematic representation of various truncation mutants containing a T7 epitope tag at the N terminus of Orc2 (MT1 to MT4). (B) IP using T7 antibody in U2OS cells expressing various mutants of T7-Orc2 and analysis of ORCA and Orc3 by immunoblotting. Note that the aa 1 to 240 fragment (MT1) associates with ORCA and that aa 227 to 577 (MT3), but not aa 452 to 577, binds to Orc3. (C) Schematic representation of smaller truncation mutants of Orc2 within the N-terminal 240 aa (MT1-1 to MT1-4) and their interaction status with ORCA. Amino acids 1 to 100 of Orc2 efficiently show ORCA binding. (D and E) Depletion of Orc2, but not Orc1, results in destabilization of cellular ORCA in human cancerous U2OS and WI38 cells. (Fa and Fb) Transient expression of MT1 (aa 1 to 240) or MT3 (aa 227 to 577) in U2OS cells depleted of endogenous Orc2 and immunoblot analysis using ORCA antibody. Note that the expression of MT1 can rescue the destabilization of ORCA. The asterisks indicate cross-reacting bands. The arrowheads indicate endogenous ORCA.

Fig 6

ORCA interacts with the C terminus of geminin and Cdt1. (A) Schematic representation of various truncation mutants containing a T7 epitope tag at the N terminus of geminin (MT-G1 to MT-G7). (B) IP in U2OS cells expressing various T7-geminin mutants using T7 antibody and analysis of Cdt1 by immunoblotting. Note that aa 80 to 160 of geminin is sufficient to bind Cdt1. (C) IP in U2OS cells expressing various T7-geminin mutants using ORCA antibody and analysis of T7-geminin by immunoblotting. Note that ORCA associates with the C-terminal end of geminin. (D) Schematic representation of various truncation mutants containing a T7 epitope tag at the N terminus of Cdt1 (MT-C1 to MT-C5). The plusses indicate the extent of interaction based on the immunoblots shown in panels E and F. (E) IP in U2OS cells expressing various T7-Cdt1 mutants using T7 antibody and analysis of geminin by immunoblotting. (F) IP in U2OS cells expressing various T7-Cdt1 mutants and hemagglutinin (HA)-ORCA using T7 antibody and analysis of ORCA by immunoblotting. Note that the aa 368 to 546 fragment of Cdt1 associates with ORCA but not with geminin. (G) IP in U2OS cells expressing T7-tagged C terminus mutants of Cdt1 and HA-ORCA using T7 antibody and analysis of ORCA by immunoblotting. Note that the aa 451 to 546 fragment of Cdt1 efficiently associates with ORCA.

Fig 7

Overexpression of geminin disrupts ORCA-Cdt1 interaction. (A) ORCA immunoprecipitation in U2OS cells transiently overexpressing T7-geminin, followed by ORCA, geminin, Orc2, and Cdt1 immunoblots. Note the loss of ORCA-Cdt1, but not ORCA-Orc2, interaction in cells overexpressing geminin. The arrowheads indicate endogenous and overexpressed geminin. (B) ORCA immunoprecipitation in U2OS cells transiently overexpressing T7 vector (VT), T7-geminin (full-length [FL]), or T7-geminin aa 1 to 160 (G2, which associates only with Cdt1, but not ORCA), followed by geminin, ORCA, Cdt1, and T7 immunoblots. Note that overexpression of full-length geminin, as well as truncated geminin, disrupts ORCA-Cdt1 binding. (C) geminin immunoprecipitation in U2OS cells transiently overexpressing T7-ORCA, followed by geminin, T7, Cdt1, and Orc2 immunoblotting. Note that the overexpression of ORCA does not affect Cdt1-geminin complex formation.

Fig 8

Model depicting ORCA association with the pre-RC components in a cell cycle-regulated manner. In late M/G₁, ORCA associates with ORC and Cdt1. Along with Cdc6, pre-RCs are assembled at origins. ORCA is abundant in the G₁ phase. One prediction would be that ORCA acts as a scaffold and facilitates the assembly of Cdt1 during M and G₁ and that of geminin at the end of G₁ on the chromatin. At the end of G₁, geminin levels begin to rise. The balance in the Cdt1-geminin ratio at this time point favors replication licensing. At the G₁/S boundary, as geminin levels begin to increase significantly and ORCA levels begin to decrease, ORCA-Cdt1 interaction is lost and geminin titrates all the Cdt1 away from ORCA, and hence from the origins. This makes the complex licensing inactive, following which Cdt1 is ubiquitinated and degraded. During G₂, the ORCA-ORC(2-5)-geminin complex prevents licensing. During mitosis, an ORCA-ORC(2-5)-phosphorylated Cdt1-phosphorylated geminin complex exists, and at the end of mitosis, geminin is degraded and Orc1 is dephosphorylated, and thus, a functional pre-RC is assembled.