Dimitrova DS , Todorov IT , Melendy T , Gilbert DM .

GM56406 NIGMS NIH HHS , GM57233-01 NIGMS NIH HHS , R01 GM057233 NIGMS NIH HHS , R29 GM056406 NIGMS NIH HHS

	Figure 1. Experimental protocol (see text for details). Representative photographs of the types of replication patterns prevailing in the early (first half, euchromatic) or late (second half, three sequential heterochromatic patterns) stages of the 10–12 h S-phase in CHOC 400 cells are displayed.
	Figure 3. PCNA does not bind nuclear components during G1-phase and associates transiently with chromosomal domains at the time when they replicate. (A) The association of PCNA (red) with the earliest-replicating chromatin regions (CldU-tagged, green) was examined in aliquots of the cells used in the experiment displayed in Fig. 2. (B) Late-replicating genomic regions were pulse-labeled with CldU (green) at 8 h in S-phase in a parallel culture of CHOC 400 cells, mitotic cells were collected few hours later, released in the subsequent cell cycle for the same periods of time and processed in the same way as in Fig. 2.
	Figure 4. RPA, but not Mcm2, is present at PCNA-containing active mammalian replication forks. Aliquots of the cell cultures (synchronized in early or mid/late S-phase) used in Fig. 2 were immunostained with PCNA-specific (red) and, either Mcm2-specific (A, green), or RPA-specific (B, green) antibodies. Shown are single sections obtained by dual-color confocal laser scanning microscopy.
	Figure 5. Variations in the amount of chromatin-bound Mcm2 and PCNA 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 extractions) and incubated for 5 min 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% (A, B, C-Mcm2, and D) or 18% (C, PCNA) SDS–polyacrylamide gels (amounts corresponding to 2 × 105 cells were loaded in each lane), transferred to nylon membranes and probed with an anti-Mcm2 antibody (A–D) or anti-PCNA antibody (C, PCNA and F). (A) Immunoblotting analysis of chromatin-bound Mcm2 at 20, 40, and 60 min after release of CHOC 400 cells from metaphase block. 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. (B) Immunoblot analysis of chromatin-bound Mcm2 at different times during interphase. The permeabilization of cells with 70 μg/ml digitonin produces nuclei with intact membranes and can be used as a reference for the total amount of nuclear Mcm2 proteins. The relative amounts of chromatin-bound Mcm2 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. (C) Western blots of total CHOC400 cellular protein extract probed with anti-Mcm2 or anti-PCNA antibodies. Positions of molecular mass standards (indicated in kD) are marked on the left of each blot. (D) Hamster Mcm2, like human BM28/Mcm2 (Todorov et al. 1995), exists as different isoforms. A lighter exposure of immunoblots probed with Mcm2-specific antibodies reveals that Mcm2 proteins can be resolved as a doublet of bands (marked with stars) in the soluble protein fraction. Only a slow moving form is detected in the nuclear pellet fraction. (E) Immunofluorescent analysis of Mcm2 chromatin-binding in Triton-extracted CHOC 400 cells at different stages of mitosis. Asynchronous cells grown on coverslips were extracted with 0.5% Triton in CSK buffer for 2 min on ice, fixed with formaldehyde, and immunostained for Mcm2 as in Fig. 2. DNA was stained with DAPI as in A. The arrowheads point to cells in prophase (i) and (iv), metaphase (ii) and (v), and late telophase (iii) and (vi). The arrows point to a cell in late anaphase/early telophase (iii) and (vi). (F) Immunoblot analysis of chromatin-bound PCNA at different times during interphase. The same protein blots used in B were probed with a PCNA-specific antibody. An increase in the total amount of cellular PCNA at the beginning of S-phase is evident, consistent with previously reported data on human PCNA (Morris and Mathews 1989). The level of chromatin-bound PCNA increased after release into S-phase and maximal amount of insoluble PCNA was detected during early S-phase.
	Figure 6. Hamster RPA does not assemble into detergent-resistant pre-RC foci during early G1-phase and associates transiently with chromosomal domains at the specific time of S-phase when they replicate. The association of RPA (red), with earliest-replicating (CldU, green, A) or late-replicating (IdU, green, B) chromatin regions was examined in aliquots of the cells used in Fig. 2. Immunolabeling was performed as in Fig. 2.
	Figure 8. Hamster RPA and PCNA are assembled into detergent-resistant replicative complexes within less than an hour from the start of S-phase. CHOC 400 cells, synchronized in mitosis, were released for 8 h in the following cell cycle (20% of the cells had entered S-phase at this time). The cells were pulse-labeled with 100 μM CldU for 1 min, extracted with Triton and fixed with formaldehyde as described in Materials and Methods. Aliquots of the cells were stained with anti-CldU antibodies (green) and either anti-RPA antibodies (red, A) or anti-PCNA antibodies (red, B). S-phase cells display yellow coloration resulting from the colocalization of RPA or PCNA (red) with nascent DNA (CldU, green). Arrowheads point to nuclei that exhibit positive staining for RPA or PCNA, but no CldU label. Arrows indicate the positions of G1-phase cells (barely visible as faint shadows).

Adachi, Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. 1992, Pubmed, Xenbase

Adachi, Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. 1992, Pubmed , Xenbase
Adachi, Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts. 1994, Pubmed , Xenbase
Aparicio, Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. 1997, Pubmed
Aten, DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication. 1992, Pubmed
Bakker, An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA. 1991, Pubmed
Bravo, Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. 1987, Pubmed
Brosh, Functional and physical interaction between WRN helicase and human replication protein A. 1999, Pubmed
Brénot-Bosc, Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells. 1995, Pubmed
Cardoso, Reversal of terminal differentiation and control of DNA replication: cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. 1993, Pubmed
Carpenter, Role for a Xenopus Orc2-related protein in controlling DNA replication. 1996, Pubmed , Xenbase
Chong, Purification of an MCM-containing complex as a component of the DNA replication licensing system. 1995, Pubmed , Xenbase
Coleman, The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. 1996, Pubmed , Xenbase
Coué, Chromatin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication. 1996, Pubmed , Xenbase
Diller, Eukaryotic replication origins: control in space and time. 1994, Pubmed
Dimitrova, Regulation of mammalian replication origin usage in Xenopus egg extract. 1998, Pubmed , Xenbase
Din, Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. 1990, Pubmed
Distel, A unified nomenclature for peroxisome biogenesis factors. 1996, Pubmed
Dutta, Initiation of DNA replication in eukaryotic cells. 1997, Pubmed
Fang, Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle. 1993, Pubmed , Xenbase
Fangman, A question of time: replication origins of eukaryotic chromosomes. 1992, Pubmed
Fry, The three faces of the WS helicase. 1998, Pubmed , Xenbase
Gilbert, Replication origins in yeast versus metazoa: separation of the haves and the have nots. 1998, Pubmed , Xenbase
Gilbert, Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure. 1995, Pubmed , Xenbase
Hamlin, Defining origins of replication in mammalian cells. 1994, Pubmed
Hozák, Visualization of replication factories attached to nucleoskeleton. 1993, Pubmed
Hozák, Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle. 1994, Pubmed
Kearsey, MCM proteins: evolution, properties, and role in DNA replication. 1998, Pubmed
Kill, The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts. 1991, Pubmed , Xenbase
Kobayashi, Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone. 1998, Pubmed
Krude, Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. 1995, Pubmed
Krude, Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication. 1996, Pubmed , Xenbase
Kubota, Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. 1995, Pubmed , Xenbase
Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 1970, Pubmed
Lawlis, Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts. 1996, Pubmed , Xenbase
Leonhardt, A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. 1992, Pubmed
Ma, Spatial and temporal dynamics of DNA replication sites in mammalian cells. 1998, Pubmed
Madine, MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. 1995, Pubmed , Xenbase
Madsen, S-phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis. A role for cyclin in DNA replication? 1985, Pubmed
Manders, Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. 1992, Pubmed
Manders, Dynamic behavior of DNA replication domains. 1996, Pubmed
Montecucco, The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication. 1995, Pubmed
Morris, Regulation of proliferating cell nuclear antigen during the cell cycle. 1989, Pubmed
Murti, Dynamics of human replication protein A subunit distribution and partitioning in the cell cycle. 1996, Pubmed
Nakayasu, Mapping replicational sites in the eucaryotic cell nucleus. 1989, Pubmed
Nethanel, Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40. 1990, Pubmed
Newlon, Putting it all together: building a prereplicative complex. 1997, Pubmed , Xenbase
O'Keefe, Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences. 1992, Pubmed
Richter, High-molecular-mass complexes of human minichromosome-maintenance proteins in mitotic cells. 1997, Pubmed
Romanowski, XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication. 1996, Pubmed , Xenbase
Romanowski, The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin. 1996, Pubmed , Xenbase
Rowles, Chromatin proteins involved in the initiation of DNA replication. 1997, Pubmed
Suzuki, Werner syndrome helicase contains a 5'-->3' exonuclease activity that digests DNA and RNA strands in DNA/DNA and RNA/DNA duplexes dependent on unwinding. 1999, Pubmed
Tanaka, Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. 1997, Pubmed
Tanaka, Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases. 1998, Pubmed
Todorov, BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. 1995, Pubmed
Tsurimoto, Simian virus 40 DNA replication in vitro: identification of multiple stages of initiation. 1989, Pubmed
Tye, MCM proteins in DNA replication. 1999, Pubmed
Wang, Lagging-strand, early-labelling, and two-dimensional gel assays suggest multiple potential initiation sites in the Chinese hamster dihydrofolate reductase origin. 1998, Pubmed
Wold, Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. 1997, Pubmed
Wu, A distinct G1 step required to specify the Chinese hamster DHFR replication origin. 1996, Pubmed , Xenbase
Yan, Replication focus-forming activity 1 and the Werner syndrome gene product. 1998, Pubmed , Xenbase
Yan, An analysis of the regulation of DNA synthesis by cdk2, Cip1, and licensing factor. 1995, Pubmed , Xenbase
Yan, FFA-1, a protein that promotes the formation of replication centers within nuclei. 1995, Pubmed , Xenbase
Zou, Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin. 1998, Pubmed