Interactions of MCP1 with components of the replication machinery in mammalian cells

Abstract

Eukaryotic DNA replication starts with the assembly of a pre-replication complex (pre-RC) at replication origins. We have previously demonstrated that Metaphase Chromosome Protein 1 (MCP1) is involved in the early events of DNA replication. Here we show that MCP1 associates with proteins that are required for the establishment of the pre-replication complex. Reciprocal immunoprecipitation analysis showed that MCP1 interacted with Cdc6, ORC2, ORC4, MCM2, MCM3 and MCM7, with Cdc45 and PCNA. Immunofluorescence studies demonstrated the co-localization of MCP1 with some of those proteins. Moreover, biochemical studies utilizing chromatin-immunoprecipitation (ChIP) revealed that MCP1 preferentially binds replication initiation sites in human cells. Interestingly, although members of the pre-RC are known to interact with some hallmarks of heterochromatin, our co-immunoprecipitation and immunofluorescence analyses showed that MCP1 did not interact and did not co-localize with heterochromatic proteins including HP1β and MetH3K9. These observations suggest that MCP1 is associated with replication factors required for the initiation of DNA replication and binds to the initiation sites in loci that replicate early in S-phase. In addition, immunological assays revealed the association of MCP1 forms with histone H1 variants and mass spectrometry analysis confirmed that MCP1 peptides share common sequences with H1.2 and H1.5 subtypes.

Keywords: DNA replication; MCP1; histone H1; interacting proteins; pre-replication complex.

Fig 1

Immunoprecipitation (IP) of HeLa nuclear extracts at different phases of the cell cycle with anti-MCP1 antibody. Western blot (WB) of HeLa nuclei from non-synchronized cells with antibodies used the detect MCP1 interacting proteins. (ES) - cells that were synchronized at early S-phase by double thymidine block (DTB); (MS) - cells at middle S-phase collected 4 hours after DTB release; (LS/G₂) - cells at late-S/G₂ phase obtained 7 hours after DTB release. As a control, HeLa nuclear extracts at G₁/S transition were immunoprecipitated with a mouse immunoglobulin (mIgG). (A) Immunoblotting with anti-MCP1 antibody detects the 33 kDa and 31 kDa forms of MCP1; (B) Proteins that interact with MCP1 were detected by western blot with antibodies against MCM2, MCM3, MCM7, ORC2, Cdc6 and ORC4; (C) Cdc45 and (D) PCNA are co-immunoprecipitated with MCP1; (E) HP1β is not associated with MCP1. (F) As a loading control a Western blot of HeLa total cell extracts at the same cell cycle phases was done with anti-βtubulin antibody.

Fig 2

(I) Cell cycle profile of K562 cells before (A) and after elutriation at G₁ (B), early-S (C), middle-S, (D), late-S (E) and G₂ (F) phases. (II) K562 nuclear extracts synchronized by elutriation at G₁, early-S (ES), middle-S (MS), and late-S (LS) were immunoprecipitated with anti-MCP1 antibody and detection of interacting proteins were done by western blot with anti-MCP1 antibody (A), that identify the 33 kDa and 31 kDa forms of MCP1, antibodies against MCM2, MCM3, MCM7, ORC2, Cdc6 and ORC4 (B), Cdc45 (C) and PCNA (D), that recognize the corresponding proteins, and a mouse IgG (mIgG) (E) was used as a control.

Fig 3

Reverse co-immunoprecipitation (IP) of HeLa nuclear extracts, synchronized at early S-phase by double thymidine block (DBT), with antibodies against Cdc6, ORC2, ORC4, MCM2, MCM3, MCM7, Cdc45, PCNA, HP1β, MetH3K9 and MCP1 followed by immunoblotting with anti-MCP1 antibody. The 31 kDa form of MCP1 is co-immunoprecipitated with all antibodies used except antibodies against HP1β and MetH3K9. Western blot (WB) immunoprecipitation (IP) with anti-MCP1 antibody recognizes the 33 kDa and 31 kDa MCP1 polypeptides in HeLa nuclear extracts synchronized at early S-phase by DBT.

Fig 4

(A) Western blot with anti-MCP1 antibody using K562 nuclear extracts that were previously immunoprecipitated with MCP1 (left), firstly depleted of MCP1 followed immunoprecipitation with ORC2 antibody (middle), and total nuclear extracts not immunoprecipitated (right). (B) Western blot with anti-ORC2 antibody using K562 nuclear extracts that were previously immunoprecipitated with MCP1 (left), initially depleted of MCP1 followed immunoprecipitation with ORC2 antibody (middle), and total nuclear extracts not immunoprecipitated (right). (C) Western blot with anti-HP1β antibody using K562 nuclear extracts that were previously immunoprecipitated with ORC2 (left), depleted of ORC2 followed immunoprecipitation with HP1β antibody (middle), and total nuclear extracts not immunoprecipitated (right).

Fig 5

(A) K562 histone preparations were immunoprecipitated (IP) with anti-MCP1, histone H1 (total), histone H1.2 and histone H1.5 antibodies followed by western blot (WB) performed with anti-MCP1 antibody. The right lane, used as a control, shows the reactivity of MCP1 with anti-MCP1 antibody (B) Immunoblotting with anti-MCP1 antibody of K562 total nuclear extracts that were firstly depleted of MCP1, H1.2 and H1.5 using the respective antibodies. (C) (I) Western blot with anti-H1.2 antibody of K562 total nuclear extracts not depleted (left) and depleted (right) of MCP1; and (II) Western blot with anti-H1.2 antibody of total histone preparations not precipitated and previously immunoprecipitated with anti-MCP1, anti-H1.2 and anti-H1.5 antibodies. (D) (I) Immunoblotting with anti-H1.5 antibody of K562 total nuclear extracts not depleted (left) and depleted of MCP1 (right) and (II) Western blot with anti-H1.5 antibody of total histone preparations not precipitated and previously immunoprecipitated with anti-MCP1, anti-H1.2, anti-H1.5 and anti-H1 (total) antibodies. (E) Western blot with anti-H1 antibody of total histone preparations not precipitated and previously immunoprecipitated with anti-MCP1, anti-H1.2 and anti-H1 antibodies.

Fig 6

Chromatin immunoprecipitation (ChIP) from K562 cells using anti-MCP1 antibody followed by real time PCR analysis of the abundance of specific DNA sequences in MCP1-bound chromatin. Probes included the human beta-globin (hBG), human lamin B2 (hLB2), human amylase (hA), human gamma-globin (hGG) and human collagen (hC). The human lamin B2 and beta-globin loci replicate early in K562 cells, and the human amylase replicate late. The human gamma-globulin and human collagen were used for a non-origin control.

Fig S1

Co-localization patterns, in non-synchro­nized cultures, in human MO59K cells of MCP1 (red) and Cdc6 (A), ORC4 (B), ORC2 (C), MCM3 (D), MCM7 (E), MCM2 (F), Cdc45 (G), HP1β (H), and MetH3K9 (I) proteins (green). See text for details. Barr = 10μc.

Fig S2

Immunolocalization of MCP1 (red) and BrdU (green) in human MO59K cells. Classification of cells in S-phase relies on the observation that cells display replication sites as small spots throughout the nucleus (A) at very early S-phase (BrdU pattern I). Later on during early S-phase (BrdU pattern II) replication foci are larger, discrete and stronger near the nuclear boundary (B). During middle S-phase (BrdU pattern III) replication sites are located at the nuclear periphery and near the perinucleolar region (C). As cells progress through S-phase, the late replication foci (BrdU pattern IV) are larger, less in number, and at few discrete sites at the periphery of the nucleus at late S-phase (D). Very late S-phase (BrdU pattern V) is characterized by the presence of smaller number and larger areas of replication sites in the interior of the nucleus and some less significant sites at the periphery of the nucleus (E). Barr = 10μc.

Fig S3

Immunolocalization of MCP1 (red) and PCNA (green) in non-synchronized human MO59K cells. MCP1 associates with PCNA at early S-phase (A) replicating foci. In middle S-phase (B) MCP1 partially co-localizes with PCNA. At late S-phase (C), MCP1 does not co-localize with PCNA. Barr = 10μc.

Fig S4

(I) Immunolocalization, of MCP1 (red) and PCNA (green) in human MO59K control cells shows that both proteins co-localize at early S-phase (A). MCP1 and PCNA do not co-localize after treatment with 1 μg/ml aphidicolin (APH) for 1 hour (B), or after 20 hours of incubation with 5 μg/ml aphidicolin (C), 2.5 mM hydroxyurea (HU) (D), or 2.5 mM thymidine (Thym) (E). (II) Immunolocalization patterns of MCP1 (red) and BrdU (green) in human MO59K cells after treatment with 1 μg/ml aphidicolin for 1 hour. MCP1 associates with BrdU replicating foci at very early (A) and early S-phases (B), at middle S-phase MCP1 partially co-localizes with BrdU (C), and at late (D) and very late S-phases, MCP1 and BrdU do not co-localize (E). Barr = 10μc.