Mammalian nuclei become licensed for DNA replication during late telophase
Daniela S. Dimitrova1,*,
,
Tatyana A. Prokhorova2,
,
J. Julian Blow2,
Ivan T. Todorov3 and
David M. Gilbert1
1 Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA
2 CRC Chromosome Replication Research Group, The Wellcome Trust Building, University of Dundee, Dundee, DD1 5EH, UK
3 Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, 1500 Duarte Road, Duarte, CA 91010, USA
* Present address: Department of Biological Sciences, Cooke Hall, North Campus, SUNY at Buffalo, Buffalo, NY 14260
Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115
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Fig. 1. Cell cycle-regulated association of hamster Mcm3 with chromatin. (A) Western blot of total CHOC 400 cellular protein extract probed with an Mcm3-specific antibody. Positions of molecular weight standards (indicated in kDa) are marked on the left. (B,C) Variations in the amount of chromatin-bound Mcm3 during different stages of the cell cycle in CHOC 400 cells. Synchronized cell populations were resuspended in cytoskeleton buffer (for Triton extractions) or transport buffer (for digitonin permeabilization) and incubated for 5 minutes on ice with or without addition of permeabilizing agents as described in Materials and Methods. The cellular or nuclear pellets (P) were separated from the soluble fractions (S) by centrifugation. The proteins from each fraction (only the pellets were analyzed in the case of digitonin extractions) were separated by electrophoresis in 10% SDS-polyacrylamide gels (amounts corresponding to 5x105 cells were loaded in each lane), transferred to nylon membranes and probed with an anti-Mcm3 antibody. In B, the percentages of cells in different stages of mitosis, determined microscopically after staining aliquots of the synchronized cells with 0.1 µg/ml 4', 6-diamidino-2-phenylindole (DAPI), are indicated on the right for each time point. In C, the relative amounts of chromatin-bound Mcm3 proteins at each time point analyzed were estimated by comparing serial dilutions of the soluble fractions run in parallel to an aliquot of the Triton-resistant fraction and are indicated on the right.
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Fig. 2. The replication capacity of CHOC 400 G1-phase nuclei is independent of the presence of XMcm3 in Xenopus egg extracts. Synchronized CHOC400 cells were permeabilized with 80 µg/ml (A,B) or 400 µg/ml (C,D) digitonin to prepare cells with intact or permeabilized nuclei, respectively. Permeabilized cells were introduced in mock-depleted (A,C) or XMcm3-depleted (B,D) Xenopus egg extracts supplemented with [ -32P]dATP. Aliquots were removed at the indicated times and the percentage of input DNA replicated was determined by acid precipitation as described (Dimitrova and Gilbert, 1998). Similar results were obtained in three independent experiments. Filled squares represent metaphase cells; diamonds, 1 hour; filled circles, 2 hours; triangles, 4 hours; hatched squares, 6-hour G1-phase nuclei; and hatched circles, G1/S-phase nuclei.
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Fig. 3. Geminin prevents binding of Xenopus Mcm proteins to hamster chromatin. Anti-HsMcm2 and anti-XMcm3 antibodies recognize both Xenopus (lane 1, Xenopus egg cytosol) and hamster (lane 2, Triton-extracted CHOC 400 G1-phase nuclei) Mcm proteins, which can be distinguished by their slightly different electrophoretic mobility. The anti-XORC2 antibody does not crossreact with hamster ORC2. (lanes 3-6) CHOC 400 metaphase cells were permeabilized with 80 µg/ml digitonin and incubated for 1 hour at 21°C in LSS (lanes 4,5) or HSS (lane 6) Xenopus egg extracts (supplemented with 100 µg/ml aphidicolin). Soluble or loosely bound proteins were removed by Triton extraction as described in Materials and Methods and the washed chromatin was subjected to immunoblotting analysis with antibodies specific for Mcm2, Mcm3 and ORC2. In lane 5, the LSS extract was supplemented with 2 µg/ml of purified Xenopus geminin. Aliquots of Xenopus egg extract (lane 1) or Triton-extracted CHOC 400 G1 nuclei (lane 2) were run in parallel and served as markers for the mobility of the respective proteins of hamster or frog origin.
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Fig. 4. Hamster chromatin becomes licensed for replication at the end of mitosis. (A-C) Geminin prevents replication of Xenopus sperm (A; 1,000 nuclei/µl) and CHO metaphase chromatin (B; 10,000 nuclei/µl), but has no effect on hamster G1 nuclei (C; 6 hours in G1, 10,000 nuclei/µl). The indicated substrates were incubated in Xenopus egg extracts with (diamonds, 0.2 µg/ml; circles, 2 µg/ml; and triangles, 20 µg/ml) or without (squares) the addition of purified geminin. Aliquots were removed at the indicated times and the amount of replicated DNA was determined by acid precipitation as in Fig. 2. (D-G) Synchronized CHOC 400 cells were permeabilized as in Fig. 2 and incubated in control (D,F) or geminin-supplemented (E,G; 2 µg/ml) Xenopus egg extracts. The percentage of replicated DNA at various time points was determined as in Fig. 2. Similar results were obtained in four independent experiments.
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Fig. 5. Replication origin selection in the DHFR locus in CHOC 400 G1 nuclei is independent of Xenopus Mcm proteins. Intact CHOC 400 pre-ODP (2 hours in G1; squares and diamonds) or post-ODP (6 hours in G1; circles and triangles) nuclei were introduced into mock-depleted (A; squares and circles), Mcm3-depleted (A; diamonds and triangles), control untreated (B; squares and circles) or geminin-supplemented (B; diamonds and triangles) Xenopus egg extracts. Specificity of initiation in the DHFR locus was determined by the ELFH assay as described in Materials and Methods.
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Fig. 6. Replication initiates with a significant degree of specificity within permeabilized CHO post-ODP nuclei incubated in licensing-deficient Xenopus egg extracts. Digitonin-permeabilized pre-ODP (2 hours in G1; triangles and hatched circles) or post-ODP (6 hours in G1; diamonds and circles) nuclei were incubated in control untreated (diamonds and triangles) or geminin-supplemented (filled circles and hatched circles) Xenopus egg extracts and the specificity of initiation within the DHFR locus was measured by the ELFH assay. Squares represent intact post-ODP nuclei incubated in control untreated egg extracts. Similar results were obtained in three independent experiments. The relative specificity of initiation at ori-ß within permeabilized post-ODP nuclei (measured as the average specificity of probes C, D, E and R, mapping to the region of peak initiation activity) varied in different experiments between 25 to 60-70% of that within intact post-ODP nuclei.
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Fig. 7. Establishment of a program for replication of mammalian nuclei. When CHOC 400 cells, blocked in metaphase with nocodazole, are released into G1-phase, nuclear assembly is completed within 1 hour [(Dimitrova and Gilbert, 1999; Dimitrova et al., 1999) and this study], coincident with the reactivation of general transcription (D.S.D., unpublished) and the licensing of chromatin for replication (this study). Within the next hour, chromosomal domains are directed to distinct and reproducible nuclear locations and by 2 hours into G1-phase (TDP, timing decision point) the temporal order for their replication in the upcoming S-phase is established (Dimitrova and Gilbert, 1999). At the ODP (5-6 hours in G1), specific chromosomal sites, where replication will initiate, are selected within the DHFR locus [(Wu and Gilbert, 1996) and this study]. Several hours later, at the G1/S transition (12-14 hours post-metaphase) the rise in S-phase promoting factor activity (S-phase cyclin-dependent kinases and dbf4/cdc7 kinase) leads to the assembly of replication factories and the firing of the earliest replication origins (Dimitrova et al., 1999). The four major events, which prepare the genome for replication, are highlighted in red.
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© The Company of Biologists Ltd 2002
