We investigated the association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei showed that a complex containing hOrc1p and hOrc2p occurs in a nucleaseresistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. We propose that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicated that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle.

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/ arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK cosedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.DNA in the nucleus is organized into a hierarchy of structures with the nucleosome as the basic building block. It has become widely accepted that modification of nucleosome structure is an important mechanism that regulates the accessibility of chromatin to DNA binding factors (1, 2).In the search for factors that change the structure of chromatin and the replicational activity of chromatin templates, we recently identified the proto-oncogene protein DEK as a candidate protein that changes the topology of DNA in chromatin in vitro (3). DEK is a 43-kDa phosphoprotein that was first isolated as part of a fusion protein expressed in a subtype of acute myeloid leukemias with (t6;9) chromosomal translocations (4). DEK was later identified as an autoimmune antigen in patients with pauciarticular onset juvenile rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases (5-7). In addition, DEK has been reported to be a site-specific DNA binding factor, which recognizes a specific DNA element in the human immunodeficiency virus enhancer (8).In a recent study, it was demonstrated that DEK associates with splicing complexes through interactions promoted by SR 1 proteins. It was shown that DEK associates with mRNA in a splicing-dependent manner, indicating that it could function to coordinate splicing with subsequent steps in gene expression (9). In addition DEK was found in a ϳ335-kDa five-component complex at a conserved position 20 -24 nucleotides upstream of exon-exon junctions (10).Our recent experiments have identified DEK as a protein that induces alterations in the superhelical density of DNA in chromatin (3). The change in topology was only observed with chromatin but not with naked DNA and depends on...

We have investigated the molecular mechanism by which the proto-oncogene protein DEK, an abundant chromatin-associated protein, changes the topology of DNA in chromatin in vitro. Band-shift assays and electron microscopy revealed that DEK induces both intraand intermolecular interactions between DNA molecules. Binding of the DEK protein introduces constrained positive supercoils both into protein-free DNA and into DNA in chromatin. The induced change in topology is reversible after removal of the DEK protein. As shown by sedimentation analysis and electron microscopy, the DEK-induced positive supercoiling causes distinct structural changes of DNA and chromatin. The observed direct effects of DEK on chromatin folding help to understand the function that this major chromatin protein performs in the nucleus.DNA in the eukaryotic nucleus is highly organized in a complex chromatin structure (reviewed in Ref. 1). The coiling of DNA by histones in nucleosomes is a barrier to the entry of proteins involved in transcription, replication, and other DNA transactions. Changes in chromatin structure as triggered by the combined action of histone acetyltransferases and histone deacetylases as well as chromatin remodeling complexes have a significant influence on the processes occurring in DNA (reviewed in Refs. 2-5). In search for factors that change the structure of chromatin and the replicative activity of chromatin templates, we have recently identified the proto-oncogene protein DEK as a candidate protein that changes the topology of DNA in chromatin in vitro (6).The DEK protein was initially identified in a fusion with the CAN nucleoporin in a subtype of acute myeloid leukemias involving chromosomal translocations (7). DEK was later identified as an autoantigen in several autoimmune diseases such as juvenile rheumatoid arthritis (8) or systemic lupus erythematosus (9). Despite these disease associations the function of the DEK protein in the cell remains elusive. There are two recent reports demonstrating that DEK could be involved in RNA metabolism. It was shown that DEK associates with splicing complexes through interactions mediated by SR proteins. DEK associates with the SRm160 splicing coactivator in vitro and remains bound to the exon product RNA after splicing. This association requires the prior formation of a spliceosome (10). In addition, DEK has been found in a five-component complex of ϳ335 kDa at a conserved position 20 -24 nucleotides upstream of exon-exon junctions in mRNAs (11).However, DEK also binds to and to metaphase chromosomes (15). In support of this, we determined that DEK is a constituent of oligonucleosomes, generated by micrococcal nuclease digestion of chromatin in isolated nuclei (16). Most of the DEK protein was released from nuclei after DNase treatment, whereas only around 10% was released after RNase treatment, indicating that the major fraction of DEK is associated with chromatin in vivo (16).Isolated DEK changes the topology of DNA in viral minichromosomes and reduces the accessibility...

Considerable progress has recently been made in describing the components of the machinery required for the initiation of eukaryotic genome replication. Much of the original work was performed studying yeast mutants that had lost the ability to perform defined steps in the progression of proliferating cells through the late G 1 phase or early S phase of the cell cycle. Genetic studies were complemented by cell biological and biochemical experiments leading to the identification of yeast DNA elements, known as ARS (autonomously replicating sequences), which serve as origins of replication, and of the sixmembered protein complex origin recognition complex (Orc) Mcm proteins occur in high copy numbers in nuclei of human cells. They appear to be either free in the nucleoplasm or bound to chromatin.Although not yet formally proven, it can be assumed that the binding of mammalian Mcm proteins to chromatin is also contingent upon the previous binding of Orc proteins and Cdc6 protein. The fraction of structure-bound mammalian Mcm proteins is highest at the beginning of S phase, but gradually decreases during progression of replication (21-23). Released nucleoplasmic Mcm proteins are found as large multiprotein complexes, composed of the six known members of the Mcm protein family. This complex has the tendency to desintegrate into more stable subcomplexes of which one is a dimer of the MCM3 protein (MCM3p) and the MCM5 protein (MCM5p), while the second is a trimer of proteins MCM4 (MCM4p), MCM6 (MCM6p), and MCM7 (MCM7p). The sixth member of the family, human protein MCM2 (MCM2p), is usually found to be loosely associated with these subcomplexes (24 -26).The present study was performed to investigate the mode of binding of Mcm proteins and ORC2p, a member of the origin recognition complex, on human chromatin. We prepared chromatin from proliferating human HeLa cells using low ionic strength conditions in the presence of the nonionic detergent Nonidet P-40. As first shown by Hancock (27), the nuclear envelope is removed by this procedure, while the general form and the ultrastructural characteristics of chromatin appear to remain largely unchanged. The maintenance of form in this structure allows biochemical manipulations such as pipetting and low speed centrifugations for a separation of structurebound from soluble nuclear material. We have used these prep-* This work was supported by Fonds der Chemischen Industrie and Deutsche Forschungs-gemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address:

The mini‐chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin‐dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki‐67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well‐defined spatio‐temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki‐67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth‐arrested, as confirmed by the absence of the Ki‐67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth‐arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki‐67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth. Copyright © 2001 John Wiley & Sons, Ltd.