Large tumor antigen (T antigen) was extracted from SV40‐infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono‐ or dimeric 4‐6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen‐specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen‐specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.

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Stat1α is a latent cytoplasmic transcription factor activated in response to interferon-γ (IFN-γ). The C-terminal 38 amino acids of Stat1α are required to trigger transcription and therefore may possibly serve as a transcription activation domain (TAD). Here we show that the C-terminus of Stat1α is an independent TAD which can interact with a specific group of nuclear proteins. Mutation of the Stat1 Ser727 and Leu724 decreases its transcriptional activity and affinity for the nuclear proteins. One of the interacting proteins was identified as MCM5, a member of the minichromosome maintenance (MCM) family involved in DNA replication. Both in vitro and in vivo interaction of Stat1α and MCM5 were demonstrated. Furthermore, the in vitro interaction required Ser727 and was enhanced by its phosphorylation. Transient overexpression of MCM5 enhanced transcriptional activation by Stat1α in a Ser727-dependent manner. Finally, changes in the level of nuclear localized MCM5 during the cell cycle correlated with the changes in transcriptional response to IFN-γ acting through Stat1α. These results strongly suggest that MCM5 is recruited through interaction with Stat1α in a Ser727-and Leu724-dependent manner to play a role in optimal transcriptional activation.

We have examined the posttranslational modification of the human chromatin protein DEK and found that DEK is phosphorylated by the protein kinase CK2 in vitro and in vivo. Phosphorylation sites were mapped by quadrupole ion trap mass spectrometry and found to be clustered in the C-terminal region of the DEK protein.Phosphorylation fluctuates during the cell cycle with a moderate peak during G 1 phase. Filter binding assays, as well as Southwestern analysis, demonstrate that phosphorylation weakens the binding of DEK to DNA. In vivo, however, phosphorylated DEK remains on chromatin. We present evidence that phosphorylated DEK is tethered to chromatin throughout the cell cycle by the un-or underphosphorylated form of DEK.The DEK protein was initially identified as a fusion protein with CAN nucleoporin in a subtype of acute myeloid leukemia involving the t(6;9) chromosomal translocation (34). Subsequently, DEK was found to be the target of autoantibodies in several diseases, including systemic lupus erythematosus (9, 10, 38), juvenile rheumatoid arthritis (10, 32), and sarcoidosis (9, 10). Interestingly, DEK has also been linked to ataxia-telangiectasia, because a fragment of DEK cDNA reverses the mutagen-sensitive phenotype of cells from ATM patients (28). Despite the number of clinical observations, the biological function of DEK remains unclear (33).Several reports suggest an involvement of DEK in transcriptional regulation. By using cofractionation and coimmunoprecipitation, it was demonstrated that the transcriptional corepressor hDaxx associates with DEK (21). However, the exact function of DEK in hDaxx-mediated repression is not clear. DEK has also been found to be associated with the latencyassociated nuclear antigen, which is constitutively expressed in Kaposi's sarcoma-associated herpesvirus latent infection (25). The data indicate that the latency-associated nuclear antigen is tethered to chromatin through its interaction with the DEK protein and the methyl CpG binding protein MeCP2. In addition, DEK interacts with cell type-specific transcription factor AP-2␣ in vitro and stimulates transactivation activity of AP-2␣ over the APOE promoter (8). Thus, DEK could be involved in linking several different proteins to chromatin. It has been reported that DEK binds to DNA and specifically recognizes the peri-ets sites in the human immunodeficiency virus type 2 enhancer (11, 14, 15) and to class II major histocompatibility complex Y-box sequences (1). However, our experiments had shown that DEK recognizes DNA structures rather than DNA sequences and preferentially binds supercoiled and four-way junction DNAs (35). These data suggest that DEK functions as an architectural protein in chromatin. Indeed, DEK is a constituent of oligonucleosomes, generated by micrococcal nuclease digestion of chromatin in isolated nuclei (22) and associates with metaphase chromosomes (13). Purified DEK changes the topology of DNA in viral minichromosomes and reduces the accessibility of chromatin to DNA binding factors including comp...

We investigated the binding regions of components of the origin recognition complex (ORC) in the human genome. For this purpose, we performed chromatin immunoprecipitation assays with antibodies against human Orc1 and Orc2 proteins. We identified a binding region for human Orc proteins 1 and 2 in a <1-kbp segment between two divergently transcribed human genes. The region is characterized by CpG tracts and a central sequence rich in AT base pairs. Both, Orc1 and Orc2 proteins are found at the intergenic region in the G 1 phase, but S-phase chromatin contains only Orc2 protein, supporting the notion that Orc1p dissociates from its binding site in the S phase. Sequences corresponding to the intergenic region are highly abundant in a fraction of nascent DNA strands, strongly suggesting that this region not only harbors the binding sites for Orc1 protein and Orc2 protein but also serves as an origin of bidirectional DNA replication.The origin recognition complex (ORC) was first identified in Saccharomyces cerevisiae as a multiprotein factor that binds to yeast origins of replication in an ATP-dependent manner (4). ORC is composed of six protein subunits, Orc1p to Orc6p, encoded by essential yeast genes whose deletions result in lethality. Components of ORC interact with the Cdc6 protein as an early step in the assembly of prereplicative complexes that are subsequently completed by the loading of Mcm protein hexamers (3,18,43,64).The binding sites for ORC in yeast DNA are known as ARS (for autonomously replicating sequences) because they direct the extrachromosomal replication of ARS-bearing plasmids. Chromosomal ARS elements serve as origins of bidirectional DNA replication in yeast and consist of about 150 bp of DNA with an essential 11-bp AT-rich ARS consensus sequence A and two or three short stimulatory sequences, B1 to B3, which are functionally important but divergent in sequence (36,40). The ORC binding site in yeast origins is a bipartite DNA sequence that includes the consensus A element and the adjacent B1 element (4, 47, 54). The B3 element is the binding site for a transcription factor, Abf1 (19). Protein-DNA crosslinking studies show that subunits Orc1p, Orc2p, Orc4p, and Orc5p contact a DNA strand in the major groove of the ARS binding site and may determine the specificity of the reaction, whereas subunits Orc3p and Orc6p appear to form proteinprotein contacts (33). Bidirectional DNA replication is initiated in the immediate neighborhood of the ARS B1 element (8), suggesting that DNA-bound ORC determines the start points for DNA replication in yeast.Proteins homologous to the yeast ORC, as well as to other proteins required for prereplicative complex formation, have been detected in all eukaryotes examined, suggesting that the manner of prereplicative complex formation may be highly conserved. Experiments with Xenopus egg extracts have clearly shown that chromatin-bound ORC serves as a landing pad for the subsequent binding of Cdc6 which, together with the Cdt1 protein, is required for the recruitment...

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.

An extrachromosomally replicating plasmid was used to investigate the specificity by which the origin recognition complex (ORC) interacts with DNA sequences in mammalian cells in vivo. We first showed that the plasmid pEPI-1 replicates semiconservatively in a once-per-cell-cycle manner and is stably transmitted over many cell generations in culture without selection. Chromatin immunoprecipitations and quantitative polymerase chain reaction analysis revealed that, in G1-phase cells, Orc1 and Orc2, as well as Mcm3, another component of the prereplication complex, are bound to multiple sites on the plasmid. These binding sites are functional because they show the Sphase-dependent dissociation of Orc1 and Mcm3 known to be characteristic for prereplication complexes in mammalian cells. In addition, we identified replicative nascent strands and showed that they correspond to many plasmid DNA regions. This work has implications for current models of replication origins in mammalian systems. It indicates that specific DNA sequences are not required for the chromatin binding of ORC in vivo. The conclusion is that epigenetic mechanisms determine the sites where mammalian DNA replication is initiated.