Proteins of the origin recognition complex (ORC) and DNA topoisomerases on mammalian chromatin.

BACKGROUND: The process of DNA replication requires the separation of complementary DNA strands. In this process, the unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. So topoisomerases play an important role in DNA replication. In order to provide more information about topoisomerases in the initiation of mammalian replication, we investigated whether topoisomerases occur close to ORC in the chromatin of cultured human HeLa cells. RESULTS: We have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC) in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5) and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin. CONCLUSION: Topoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.

The distribution of the DEK protein in mammalian chromatin.

DEK is an abundant and ubiquitous chromatin protein. Here we investigate whether DEK is regularly distributed in the chromatin of human HeLa cells. We show that DEK appears to be excluded from the heterochromatic compartment. However, DEK seems to colocalize with a subfraction of chromatin bearing acetylated histone H4. We examined certain DNA sequences in specifically immunoprecipitated chromatin for four selected human genes. We found that most of the investigated gene sequences were moderately enriched in immunoprecipitated chromatin. In contrast, a promoter-proximal element of the human TOP1 gene was highly enriched in the chromatin immunoprecipitates. This enrichment was lost when cells were treated with alpha-amanitin showing that DEK binds to this particular site only when the TOP1 gene is actively expressed. Our conclusion is that DEK could serve as an architectural protein at the promoter or enhancer sites of a subfraction of human genes.

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform.

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.

DNA replication in protein extracts from human cells requires ORC and Mcm proteins.

We used protein extracts from proliferating human HeLa cells to support plasmid DNA replication in vitro. An extract with soluble nuclear proteins contains the major replicative chain elongation functions, whereas a high salt extract from isolated nuclei contains the proteins for initiation. Among the initiator proteins active in vitro are the origin recognition complex (ORC) and Mcm proteins. Recombinant Orc1 protein stimulates in vitro replication presumably in place of endogenous Orc1 that is known to be present in suboptimal amounts in HeLa cell nuclei. Partially purified endogenous ORC, but not recombinant ORC, is able to rescue immunodepleted nuclear extracts. Plasmid replication in the in vitro replication system is slow and of limited efficiency but robust enough to serve as a basis to investigate the formation of functional pre-replication complexes under biochemically defined conditions.

Distribution of the chromatin protein DEK distinguishes active and inactive CD21/CR2 gene in pre- and mature B lymphocytes.

DEK is an abundant and ubiquitous chromatin protein that has only recently attracted attention. DEK preferentially binds to cruciform and superhelical DNA and induces positive supercoils into closed circular DNA. It is quite likely therefore that DEK performs an important architectural function in chromatin. However, it is not known how DEK is distributed in chromatin. As the first study of its kind, we investigate the distribution of DEK at the CD21/complement receptor 2 gene regulatory regions in two B lymphocyte lines, namely Ramos, which expresses the CD21 gene, and Nalm-6, which does not. We use a chromatin immunoprecipitation approach and show that DEK appears to be distributed over various regions of the expressed and silent genes, but occurs in 2- to 3-fold higher amounts at a promoter-proximal site of the expressed gene. Moreover, induction of CD21 expression in Nalm-6 cells leads to accumulation of DEK at this site. We propose that the accumulation of DEK is functionally linked to gene expression.

The SAF-box domain of chromatin protein DEK.

DEK is an abundant chromatin protein in metazoans reaching copy numbers of several millions/nucleus. Previous work has shown that human DEK, a protein of 375 amino acids, has two functional DNA-binding domains, of which one resides in a central part of the molecule and contains sequences corresponding to the scaffold attachment factor-box (SAF-box) domain as found in a growing number of nuclear proteins. Isolated SAF-box peptides (amino acids 137-187) bind weakly to DNA in solution, but when many SAF-box peptides are brought into close proximity on the surface of Sephadex beads, cooperative effects lead to a high affinity to DNA. Furthermore, a peptide (amino acids 87-187) that includes a sequence on the N-terminal side of the SAF-box binds efficiently to DNA. This peptide prefers four-way junction DNA over straight DNA and induces supercoils in relaxed circular DNA just like the full-length DEK. Interestingly, however, the 87-187 amino acid peptide introduces negative supercoils in contrast to the full-length DEK, which is known to introduce positive supercoils. We found that two adjacent regions (amino acids 68-87 and 187-250) are necessary for the formation of positive supercoils. Our data contribute to the ongoing characterization of the abundant and ubiquitous DEK chromatin protein.

Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study.

IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells.

The DEK protein--an abundant and ubiquitous constituent of mammalian chromatin.

The protein DEK is an abundant and ubiquitous chromatin protein in multicellular organisms (not in yeast). It is expressed in more than a million copies/nucleus of rapidly proliferating mammalian cells. DEK has two DNA binding modules of which one includes a SAP box, a sequence motif that DEK shares with a number of other chromatin proteins. DEK has no apparent affinity to specific DNA sequences, but preferentially binds to superhelical and cruciform DNA, and induces positive supercoils into closed circular DNA. The available evidence strongly suggests that DEK could function as an architectural protein in chromatin comparable to the better known classic architectural chromatin proteins, the high-mobility group or HMG proteins.

The replicative regulator protein geminin on chromatin in the HeLa cell cycle.

Geminin is believed to have a major function in the regulation of genome replication and cell proliferation. Published evidence shows that geminin specifically interacts with Cdt1 to block its function in the assembly of prereplication complexes. However, in proliferating HeLa cells geminin and Cdt1 are co-expressed during a relatively short time at the G(1)-to-S phase transition. Under these conditions, nearly all Cdt1 and a major part of geminin are bound to chromatin and reside at the same or closely adjacent sites as shown here by chromatin immunoprecipitation. Cdt1 is rapidly degraded early in S phase, but geminin remains bound to the chromatin sites. One function that chromatin-bound geminin could perform is to prevent access to Cdt1 that may escape S phase-dependent degradation or is synthesized in excess. Indeed, Cdt1 continues to be synthesized in HeLa cells in S phase but never accumulates because of the efficient degradation. Therefore, geminin can be eliminated by RNA interference without detectable effects on cell cycle parameters.

Phosphorylation by protein kinase CK2 changes the DNA binding properties of the human chromatin protein DEK.

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.

Protein kinase CK2 phosphorylates the cell cycle regulatory protein Geminin.

Geminin contributes to cell cycle regulation by a timely inhibition of Cdt1p, the loading factor required for the assembly of pre-replication complexes. Geminin is expressed during S and G2 phase of the HeLa cell cycle and phosphorylated soon after its synthesis. We show here that Geminin is an excellent substrate for protein kinase CK2 in vitro; and that the highly specific CK2 inhibitor tetrabromobenzotriazole (TBB) blocks the phosphorylation of Geminin in HeLa protein extracts and HeLa cells in vivo. The sites of CK2 phosphorylation are located in the carboxyterminal region of Geminin, which carries several consensus sequence motifs for CK2. We also show that a minor phosphorylating activity in protein extracts can be attributed to glycogen synthase kinase 3 (GSK3), which most likely targets a central peptide in Geminin. Treatment of HeLa cells with TBB does not interfere with the ability of Geminin to interact with the loading factor Cdt1.

An episomal mammalian replicon: sequence-independent binding of the origin recognition complex.

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 S-phase-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.

Expression and phosphorylation of the replication regulator protein geminin.

It has been described that the replication regulator protein geminin is rapidly degraded at the end of mitosis and newly expressed at the beginning of the next S phase in the metazoan cell cycle. We have performed experiments to investigate the synthesis of geminin in cycling human HeLa cells. The levels of geminin-mRNA vary only modestly during the cell cycle with a 2-3-fold higher mRNA level at the G1/S phase transition, whereas newly synthesized geminin can only be detected in post-G1 phases. Surprisingly, geminin, once synthesized, does not remain stable, but is turned over during S phase with a half-life of 3-4h. We also show that geminin becomes phosphorylated as S phase proceeds and identify by MALDI mass spectrometry two specific major phosphorylation sites.

Identification and functional characterization of a new member of the human Mcm protein family: hMcm8.

The six minichromosome maintenance proteins (Mcm2-7) are required for both the initiation and elongation of chromosomal DNA, ensuring that DNA replication takes place once, and only once, during the S phase. Here we report on the cloning of a new human Mcm gene (hMcm8) and on characterisation of its protein product. The hMcm8 gene contains the central Mcm domain conserved in the Mcm2-7 gene family, and is expressed in a range of cell lines and human tissues. hMcm8 mRNA accumulates during G(1)/S phase, while hMcm8 protein is detectable throughout the cell cycle. Immunoprecipitation-based studies did not reveal any participation of hMcm8 in the Mcm3/5 and Mcm2/4/6/7 subcomplexes. hMcm8 localises to the nucleus, although it is devoid of a nuclear localisation signal, suggesting that it binds to a nuclear protein. In the nucleus, the hMcm8 structure-bound fraction is detectable in S, but not in G(2)/M, phase, as for hMcm3. However, unlike hMcm3, the hMcm8 structure-bound fraction is not detectable in G(1) phase. Overall, our data identify a new Mcm protein, which does not form part of the Mcm2-7 complex and which is only structure-bound during S phase, thus suggesting its specific role in DNA replication.

Comparisons of two large phaeoviral genomes and evolutionary implications.

The evolution of viral genomes has recently attracted considerable attention. We compare the sequences of two large viral genomes, EsV-1 and FirrV-1, belonging to the family of phaeoviruses which infect different species of marine brown algae. Although their genomes differ substantially in size, these viruses share similar morphologies and similar latent infection cycles. In fact, sequence comparisons show that the viruses have more than 60% of their genes in common. However, the order of genes is completely different in the two genomes, suggesting that extensive recombinational events in addition to several large deletions had occurred during the separate evolutionary routes from a common ancestor. We investigated genes encoding components of signal transduction pathways and genes encoding replicative functions in more detail. We found that the two genomes possess different, although overlapping, sets of genes in both classes, suggesting that different genes from each class were lost, perhaps randomly, after the separate evolution from an ancestral genome. Random loss would also account for the fact that more than one-third of the genes in one viral genome has no counterparts in the other genome. We speculate that the ancestral genome belonged to a cellular organism that had once invaded a primordial brown algal host.

Human Mcm proteins at a replication origin during the G1 to S phase transition.

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p-Orc6p). While the order of assembly--Mcm binding follows ORC binding--seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G1 phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system.

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G1 phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G1 phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G1 phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.

The origin recognition complex marks a replication origin in the human TOP1 gene promoter.

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.

Synthesis and turn-over of the replicative Cdc6 protein during the HeLa cell cycle.

The human replication protein Cdc6p is translocated from its chromatin sites to the cytoplasm during the replication phase (S phase) of the cell cycle. However, the amounts of Cdc6p on chromatin remain high during S phase implying either that displaced Cdc6p can rebind to chromatin, or that Cdc6p is synthesized de novo. We have performed metabolic labeling experiments and determined that [35S]methionine is incorporated into Cdc6p at similar rates during the G1 phase and the S phase of the cell cycle. Newly synthesized Cdc6p associates with chromatin. Pulse-chase experiments show that chromatin-bound newly synthesized Cdc6p has a half life of 2-4 h. The results indicate that, once bound to chromatin, pulse-labeled new Cdc6p behaves just as old Cdc6p: it dissociates and eventually disappears from the nucleus. The data suggest a surprisingly dynamic behaviour of Cdc6p in the HeLa cell cycle.

Identification of a binding region for human origin recognition complex proteins 1 and 2 that coincides with an origin of DNA replication.

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.