The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase

Abstract

Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

Figure 1

Comparison of Mcm10 proteins of different species. (a) Schematic diagram of the similarities among human (Homo sapiens), D.melanogaster, C.elegans, S.pombe and S.cerevisiae Mcm10 proteins. Striped boxes indicate the conserved central domain. Black boxes and CCCH indicate the zinc finger-like motif (CX_9-10-CX₁₁-CX₂H). The amino acid sequence of the human Mcm10 central domain (200 residues between 236 and 435) showed 42% identity to the D.melanogaster protein, 29% identity to the C.elegans protein, and 24% identity to the central domains of both S.pombe and S.cerevisiae proteins, respectively. Numbers at the right of the open boxes represent the amino acid residue number. The DNA sequence of the human Mcm10 gene is available from the DDBJ/GenBank/EMBL database with the accession number AB042719. (b) Multiple sequence alignment of the central domain of the above sequences using the ClustalW program. Identical residues are indicated by dark gray boxes and bold type, and similar residues are indicated by light gray boxes. The zinc finger-like motif is indicated by circles. Numbers indicate the amino acid residues.

Figure 2

Expression of human Mcm10 mRNA after the growth stimulation. After cultured in DMEM containing 0.4% fetal calf serum for 72 h, NB1–RGB cells were released by changing the medium to DMEM containing 15% fetal calf serum. Two micrograms of poly(A)⁺ RNA was isolated at the indicated times and used for northern blotting. (a) The membrane blot was probed with human Mcm10 (HsMcm10), PCNA and EF1α cDNA probes. (b) The intensity of radioactivity was measured by BAS2500, and the results are presented as percentage of maximum. DNA synthesis was monitored by BrdU incorporation. Open and closed circles represent HsMcm10 and PCNA mRNA, respectively, and triangles represent the amount of incorporated BrdU. EF1α was used as a reference of the amount of RNA loaded.

Figure 3

Localization of ectopically expressed HsMcm10 in COS-1 cells. (a) COS-1 cells were transiently transfected with the expression vector encoding the six-His-tagged HsMcm10 protein. Cells were fixed at 24 h after transfection, and the expression was detected by immunostaining with the monoclonal anti-six-His antibody and FITC-conjugated secondary antibody. Nuclear DNA was stained with 1 µg/ml of Hoechst 33258. (b) Twenty-four hours after transfection, cells were either fixed immediately or extracted with 0.5% Triton X-100 at 4°C for 10 min prior to fixation.

Figure 4

Subcellular distribution of endogenous HsMcm10 in HeLa cells. (a) Detection of HsMcm10 protein with a polyclonal antibody. Whole cell lysates from 2 × 10⁴ HeLa cells were subjected to immunoblotting (lane 1). The arrow indicates the endogenous HsMcm10. The minor additional band positioned at 88 kDa indicated by an asterisk seems to be a degradation product of HsMcm10 protein. Whole cell lysates from 2 × 10³ or 2 × 10⁴ HeLa cells overexpressing HA-epitope tagged HsMcm10 were subjected to immunoblotting (lanes 2 and 3). The arrowhead indicates the HA–HsMcm10. The minor band positioned at 91 kDa indicated by a dot seems to be a degradation product of HA–HsMcm10. (b) Whole cell extracts (WCE), Triton X-100-soluble fractions (sup), chromatin-bound fractions solubilized with DNaseI digestion (DNaseI) and DNaseI-unextractable fractions (ppt) were prepared. HeLa cells were synchronized at M phase with nocodazole treatment. HeLa cells were arrested at G₁/S boundary with aphidicolin treatment after replating mitotic cells. Each fraction from 1 × 10⁵ cells was analyzed by immunoblotting. (c) HeLa cells were released from G₁/S boundary. Cell extracts were prepared at indicated times and subjected to immunoblotting. (d) To monitor S phase, HeLa cells were pulse-labeled at the indicated times with BrdU, which was detected by fluorescent immunostaining.

Figure 5

Co-localization of ectopically expressed HsMcm10 and Orc2 in COS-1 cells. The expression vector encoding the His-tagged HsMcm10 protein was co-transfected with either the vector expressing the HA-tagged Orc2 (a) or that expressing the GFP-tagged lamin A mutant (b). Cells were fixed at 24 h after transfection, and the six-His-tagged HsMcm10 protein was detected using the monoclonal anti-six-His antibody and the Texas red-conjugated secondary antibody. HA-tagged HsMcm10 and Orc2 were detected with the polyclonal anti-HA antibody and the FITC- or Texas red-conjugated secondary antibody.

Figure 6

Two-hybrid interactions between human Mcm10 (HsMCM10), human Orc2 (HsORC), and mouse Mcm2–7 (mMCM2–7). Interactions between pairs of fusion proteins containing either an N-terminal LexA DNA-binding domain (BD) or a B42 transactivation domain (AD) were tested in the yeast two-hybrid assay using the lacZ reporter gene. The reporter β-galactosidase activity was measured using chlorophenol red-β-d-galactopyranoside (CPRG) as the substrate. Each bar represents three independent measurements. Error bars indicate standard deviation. As a positive control, plasmids containing simian virus 40 large T antigen and p53 were used. As a negative control, BD and AD vectors containing no inserts were transfected.

Figure 7

Immunoprecipitation assay with His–HsMcm10 and HA–Orc2. Fifty micrograms of the chromatin-bound fraction containing vector control (lanes 5 and 9), HA–Orc2 (lanes 6 and 10), coexpressed His–HsMcm10 and HA–Orc2 (lanes 7 and 11), or His–HsMcm10 (lanes 8 and 12) were immunoprecipitated with anti-HsMcm10 antibody (lanes 5–8) or anti-HA antibody (lanes 9–12). Precipitates were subjected to western blot analysis using anti-HsMcm10 (upper panels) and anti-HA (lower panels) antibodies. Lanes 1–4 contain 10 µg of the chromatin-bound fractions as input proteins.