Involvement of human MCM8 in prereplication complex assembly by recruiting hcdc6 to chromatin

Abstract

The MCM2-MCM7 complex is an essential component of the prereplication complex (pre-RC), which is recruited by the cdc6 and cdt1 proteins to origins of DNA replication during G(1) phase. Here, we report that the accumulation on chromatin of another member of the MCM protein family, human MCM8 (hMCM8), occurs during early G(1) phase, before the hMCM2-hMCM7 complex binds. hMCM8 interacts in vivo with two components of the pre-RC, namely, hcdc6 and hORC2. Depletion of endogenous hMCM8 protein by RNA interference leads to a delay of entry into S phase, suggesting a role for hMCM8 in G(1) progression. Furthermore, down-regulation of hMCM8 also leads to a reduced loading of hcdc6 and the hMCM2-hMCM7 complex on chromatin. These results suggest that hMCM8 is a crucial component of the pre-RC and that the interaction between hMCM8 and hcdc6 is required for pre-RC assembly.

FIG. 1.

Characterization of polyclonal antibodies generated against hMCM8. (A) Total cell extracts of HeLa, 293T (untransfected or transfected with hMCM8), and HS68 cells were subjected to SDS-PAGE, and proteins were transferred to nitrocellulose. After protein staining with Ponceau-S red (lower panel), the blot was cut and immunoblotted with either the C-terminal antibody [MCM8(741-756)], the N-terminal antibody [MCM8(1-92)], or the corresponding preimmune sera (PI) (upper panel). (B) Increasing amounts of recombinant His₆-tagged hMCM8 (200, 400, and 800 ng) were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the polyclonal antibodies anti-hMCM8(1-92) and anti-hMCM8(741-756). Molecular mass markers (in kilodaltons) are noted at the left.

FIG. 2.

hMCM8 is chromatin bound, and its chromatin binding is cell cycle regulated. (A) Asynchronous HeLa cells were subjected to a biochemical fractionation. In brief, cells were lysed with Triton X-100 in a sucrose-rich buffer. Cytoplasmic proteins (S2) were separated from the nuclei by low-speed centrifugation. Nuclei were washed and then lysed in a no-salt buffer. A second centrifugation step separated the remaining soluble nuclear proteins (S3) from an insoluble fraction (P3). Proteins found in the final pellet (P3) were likely to be bound to chromatin or the nuclear matrix. The distributions of different proteins in the total cell extract (TCE), soluble fraction (S2), solubilized nuclear proteins (S3), and chromatin-enriched fraction (P3), with or without micrococcal nuclease treatment (+ or −), are shown. (B) Chromatin association of hMCM8, the MCM2-MCM7 complex, hcdc6, and hORC2 during the cell cycle. HeLa cells were arrested at different stages of the cell cycle, the DNA content was determined by flow cytometry (upper panel), and an aliquot of cells was subjected to biochemical fractionation. Immunoblots of the soluble and insoluble fractions are shown (lower panel). The total cell extract of exponentially growing cells (TCE exp) was used as the input control. (C) Chromatin association of hMCM8 at the M- to G₁-phase transition. HeLa cells were synchronized at prometaphase with nocodazole and then harvested by mitotic shake-off. Cells were collected at different time points after release from the block and subjected to biochemical fractionation (lower panel). The DNA contents of cells harvested at the indicated time points were determined by flow cytometry (upper panel).

FIG. 2.

hMCM8 is chromatin bound, and its chromatin binding is cell cycle regulated. (A) Asynchronous HeLa cells were subjected to a biochemical fractionation. In brief, cells were lysed with Triton X-100 in a sucrose-rich buffer. Cytoplasmic proteins (S2) were separated from the nuclei by low-speed centrifugation. Nuclei were washed and then lysed in a no-salt buffer. A second centrifugation step separated the remaining soluble nuclear proteins (S3) from an insoluble fraction (P3). Proteins found in the final pellet (P3) were likely to be bound to chromatin or the nuclear matrix. The distributions of different proteins in the total cell extract (TCE), soluble fraction (S2), solubilized nuclear proteins (S3), and chromatin-enriched fraction (P3), with or without micrococcal nuclease treatment (+ or −), are shown. (B) Chromatin association of hMCM8, the MCM2-MCM7 complex, hcdc6, and hORC2 during the cell cycle. HeLa cells were arrested at different stages of the cell cycle, the DNA content was determined by flow cytometry (upper panel), and an aliquot of cells was subjected to biochemical fractionation. Immunoblots of the soluble and insoluble fractions are shown (lower panel). The total cell extract of exponentially growing cells (TCE exp) was used as the input control. (C) Chromatin association of hMCM8 at the M- to G₁-phase transition. HeLa cells were synchronized at prometaphase with nocodazole and then harvested by mitotic shake-off. Cells were collected at different time points after release from the block and subjected to biochemical fractionation (lower panel). The DNA contents of cells harvested at the indicated time points were determined by flow cytometry (upper panel).

FIG. 3.

hMCM8 interacts with hcdc6 and hORC2 but not with the hMCM2-hMCM7 complex. (A) 293T cells were transfected with either HA-tagged hMCM8 (lanes 3 and 7), HA-tagged hcdc6 (lane 4), or HA-tagged ORC2 (lane 8). Untransfected 293T cells served as negative controls (lanes 2 and 6). Total cell extract of untransfected 293T cells (TCE) was used as the input control (lanes 1 and 5). Untransfected and transfected cells were incubated with anti-HA antibodies (α-HA) overnight. Immunoprecipitates (IP) were subjected to SDS-PAGE, and immunoblotting was carried out with the indicated antibodies. (B) HeLa cells were lysed and incubated with anti-MCM8(741-756) antibodies (MCM8) or anti-MCM8(741-756) antibodies preincubated with the corresponding antigenic peptide (MCM8+pep). Immunoprecipitates were subjected to SDS-PAGE and probed in immunoblots with the indicated antibodies.

FIG. 3.

hMCM8 interacts with hcdc6 and hORC2 but not with the hMCM2-hMCM7 complex. (A) 293T cells were transfected with either HA-tagged hMCM8 (lanes 3 and 7), HA-tagged hcdc6 (lane 4), or HA-tagged ORC2 (lane 8). Untransfected 293T cells served as negative controls (lanes 2 and 6). Total cell extract of untransfected 293T cells (TCE) was used as the input control (lanes 1 and 5). Untransfected and transfected cells were incubated with anti-HA antibodies (α-HA) overnight. Immunoprecipitates (IP) were subjected to SDS-PAGE, and immunoblotting was carried out with the indicated antibodies. (B) HeLa cells were lysed and incubated with anti-MCM8(741-756) antibodies (MCM8) or anti-MCM8(741-756) antibodies preincubated with the corresponding antigenic peptide (MCM8+pep). Immunoprecipitates were subjected to SDS-PAGE and probed in immunoblots with the indicated antibodies.

FIG. 4.

Down-regulation of hMCM8 by RNAi impairs the G₁-to-S transition. (A) HeLa cells were transfected with either pS-MCM8 or pS-GL2, and the levels of hMCM8 were determined by immunoblotting at the indicated time points after transfection. (B) RNAi-mediated down-regulation of hMCM8 delays the G₁-to-S transition. HeLa cells were transfected with either pS-GL2 or pS-MCM8, treated with nocodazole for 12 h at 60 h after transfection to prevent reentry of the transfected cells into G₁, and analyzed by flow cytometry. (C) Down-regulation of hMCM8 leads to a delay in entry into S phase. HeLa cells were transfected with either pS-GL2 or pS-MCM8 and treated for 12 h with nocodazole. At 72 h after transfection, cells were harvested by mitotic shake-off, washed five times, and reseeded into fresh medium. Samples were taken at the indicated time points after release from the nocodazole block, and the DNA contents were determined by flow cytometry.

FIG. 5.

Loading of hcdc6 and hMCM proteins on chromatin is reduced after the down-regulation of hMCM8. Cells were transfected with pS-GL2 or pS-MCM8, harvested 48 (lane 1), 60 (lane 2), and 72 (lane 3) h later, and subjected to the biochemical-fractionation method as described in the legend to Fig. 2. Immunoblotting was carried out for total cell extract (TCE) (left panel) and the chromatin fraction (P3) (right panel) with antibodies against the indicated proteins. MEK1/MEK2, a cytosolic protein kinase, was used as a control.

FIG. 6.

Model describing the function of MCM8 as a loading factor for cdc6 to chromatin in human cells. See the text for details.