Nuclear Organization of DNA Replication Initiation Proteins in Mammalian Cells

Abstract

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.It has been suggested that nucleic acid metabolism is carried out on spatially organized domain structures in the cell nucleus (reviewed in Ref. 1). Non-chromatin nuclear structures such as the nuclear matrix, the scaffold, and the nucleoskeleton have been suggested as key players in organizing high order chromatin/nuclear structures (reviewed in Refs. 2 and 3). In the case of DNA replication, for example, fluorescence microscopic analyses have revealed discrete granular sites of replication, i.e. replication sites or replication foci (4 -7). Replication foci may be constructed based on non-chromatin nuclear structures, since nascent DNA and many proteins involved in DNA synthesis have been found to attach to these (Refs. 8 and 9; also reviewed in Refs. 2 and 3). In addition, non-chromatin nuclear elements have also been implicated in the initiation step of DNA replication (reviewed in Ref. 10); replication origins are often situated close to matrix/scaffold attachment regions (11)(12)(13)(14). In this regard, it is of interest to ascertain how replication initiation proteins such as the origin recognition complex (ORC), 1 CDC6, and MCM interact with non-chromatin nuclear structures.The ORC, consisting of six subunits ORC1 to -6, was originally identified as binding specifically to definitive origin sequences in budding yeast (15). Subsequent studies with yeast and Xenopus egg extracts suggested stepwise assembly of ORC, CDC6, and MCM, a six-member protein complex, onto chromatin to form prereplication complexes (reviewed in Refs. 16 and 17). An additional protein(s) is also required for the prereplication complex formation (18,19). However, the mode of...

In Vivo Dynamics of EBNA1-oriP Interaction during Latent and Lytic Replication of Epstein-Barr Virus

“…In our previous studies, chromatin was prepared by isolating a nuclear pellet from cytoplasmic and soluble fractions in the presence of Triton X-100 or an equivalent detergent (29,31). Such detergents are known to disrupt the nuclear envelope and, in addition, lead to solubilization and extraction of nuclear proteins (32).…”

“…We have previously revealed subcellular dynamics of human chromosomal DNA replication initiation proteins such as the origin recognition complex, CDC6, and the mini-chromosome maintenance (MCM) protein complex in HeLa cells using biochemical fractionation methods (29,31). We applied this method to the Tet-BZLF1/ B95-8 cells.…”

“…It was observed that cellular membranes disappeared, but nuclei remained intact morphologically as judged by microscopic analyses (data not shown). The proteins remaining in the extracted nuclei have been shown to indeed bind to the chromatin/nuclear matrix and thus represent functionally active fractions (29,31). Second, the extracted nuclei were digested with DNase I to remove the bulk of the host and EBV genomes.…”

Cell Cycle-controlled Interaction of Nucleolin with the Retinoblastoma Protein and Cancerous Cell Transformation

“…Chemical cross-linking was performed with the membranepermeable, cleavable protein-protein cross-linking reagent dithiobis-succinimidyl propionate (DSP) (Pierce), following the manufacturer's protocols and published procedures (32). The treatment of intact cells with 0.5 mM DSP for 15 min at room temperature in phosphate-buffered saline was followed by quenching with 10 mM Tris, pH 7.5.…”

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

“…40 In the Triton-extracted, DNase-treated G 1 -phase nuclei, Cdc6 has punctate nuclear staining that only partially overlaps with ORC1, indicating that at least a subset of nuclear Cdc6 is not localized to replication origins. 41 Despite questions regarding the subnuclear localization, the observation that endogenous Cdc6 is in the nucleus during S phase led to the suggestion that Cdc6 nuclear export is not important for controlling replication licensing; 39 and this uncertainty is reflected in recent reviews. [1][2][3] An alternative potential mechanism for the regulation of Cdc6 activity during S phase is suggested by results with a cell-free system in which mouse 3T3 nuclei are incubated in cell extracts.…”