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Biomedical subjects

R Knippers

Publications and source records attributed to R Knippers.

At least 37 records · Page 2Linked to original sources
PubMed1998

Stability of the replicative Mcm3 protein in proliferating and differentiating human cells.

Mcm proteins are abundant nuclear proteins involved in the regulation of genome replication. Previous experiments had shown that levels of Mcm-specific mRNAs increase at the G1/S phase transition of the cell cycle, but that the amounts of Mcm proteins do not change much during the cell cycle. To learn more about the stability of an Mcm protein we performed experiments which showed that: (i) more than 60% of [35S]methionine pulse-labeled Mcm3 protein appears to be degraded during a 24-h chase in HeLa cells; (ii) the amount of Mcm3 protein significantly decreases during the differentiation of HL60 cells in vitro (whereas another replication-initiation protein, hOrc2, remains fairly constant); and (iii) according to immunohistochemical staining, Mcm3 protein is present in nuclei of cells in the proliferating zone of human epidermal tissue, but in decreasing amounts in nuclei of differentiating cells of the upper cell layers. Our interpretation is that Mcm3 protein is no longer synthesized after initiation of differentiation and slowly disappears at a half-life of approximately 24 h.

Cell Cycle Proteins↗
PubMed1998

Human protein MCM6 on HeLa cell chromatin.

Minichromosome maintenance (Mcm) proteins perform essential functions regulating the replication of chromatin. Human cells, like other eukaryotic cells, express at least six Mcm proteins conserved in the central region. We have earlier described the primary structures of five human Mcm proteins, but the primary structure of the sixth human Mcm protein, MCM6, was identified only recently. We now use antibodies, specific for the MCM6 protein, to assess its intranuclear distribution. We find that a fraction of MCM6 protein occurs in the nucleosol, forming multiprotein complexes with other Mcm proteins. More importantly, we use for the first time micrococcal nuclease as a tool to investigate the association of MCM6 protein with chromatin. After short digestion times, a considerable fraction of the MCM6 protein is released from chromatin as a multiprotein complex that includes other Mcm proteins as well. In addition, fractions of MCM3 and MCM6 proteins are released by nuclease digestion as monomeric proteins indicating that at least these two Mcm proteins may also occur as single molecules on chromatin. The data also suggest that the chromatin regions with bound Mcm proteins are more vulnerable to nuclease attack than bulk chromatin and may therefore differ in the arrangement of nucleosomes.

Antibodies↗
PubMed1998

Selective proteolysis of the nuclear replication factor MCM3 in apoptosis.

Cleavage of specific protein subsets is a key event in the execution of apoptosis. Protein degradation may serve for the structural alterations that result in cell self-destruction, but it may also function as a switch in the decisions between apoptosis and necrosis or apoptosis and cell proliferation. Here, we show that MCM3, but not other members of the Mcm family of replicative proteins, is cleaved early in several models of apoptosis. Cleavage of MCM3 can be prevented by caspase inhibitors, and it does not occur when cells are forced to undergo necrosis by energy deprivation. We propose that active destruction of MCM3 inactivates the Mcm complex and serves to prevent untimely DNA replication events during the execution of the cell death program.

Apoptosis↗
PubMed1997

High-molecular-mass complexes of human minichromosome-maintenance proteins in mitotic cells.

Minichromosome-maintenance (Mcm) proteins perform essential functions regulating the replication of eukaryotic genomes. In interphase cells they are either bound to a nuclear structure, most probably chromatin, or occur as free multiprotein complexes in the nucleoplasm. Mcm proteins are displaced from their chromatin sites during S phase, and several become highly phosphorylated during mitosis. We investigated whether phosphorylation affects the ability of mitotic Mcm proteins to form multiprotein complexes. Our results clearly show that phosphorylated mitotic Mcm proteins form a 14-15-S complex, probably consisting of one molecule each of the six known human Mcm proteins.

Amino Acid Sequence↗
PubMed1997

Effects of bifunctional netropsin-related minor groove-binding ligands on mammalian type I DNA topoisomerase.

We investigated the effects of compounds with two covalently linked netropsin moieties (bis-netropsin) on the function of mammalian type I DNA topoisomerase (topo I) in vitro. We initiated these studies because earlier studies had shown that certain bis-netropsins possess a several-fold higher antitumor and antiviral activity than netropsin. We confirmed that the parent compound netropsin, but not its bifunctional derivatives, induce supercoils in closed DNA. We determined that bis-netropsins inhibit the binding of topo I to DNA more efficiently than netropsin and that bis-netropsins but not netropsin induce specific DNA strand cleavage in the presence of topo I. We discuss a model explaining the different effects of netropsin and bis-netropsins on topo I.

Antineoplastic Agents↗
PubMed1997

MCM4 and PRKDC, human genes encoding proteins MCM4 and DNA-PKcs, are close neighbours located on chromosome 8q12-->q13.

A human genomic DNA fragment containing the 5' region of MCM4, a gene encoding replication protein MCM4/Cdc21 was isolated. At a distance of about 800 base pairs upstream of MCM4, the fragment was shown to also contain the 5' end of PRKDC, a gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The genomic DNA fragment was used for hybridization to human metaphase chromosome spreads. The cytogenetic map location of the two closely adjacent genes was determined to be 8q12-->q13.

Cell Cycle Proteins↗
PubMed1996

A novel human Mcm protein: homology to the yeast replication protein Mis5 and chromosomal location.

Mcm proteins perform functions related to the regulation of eukaryotic genome replication. Previous work has shown that human cells contain at least five different Mcm proteins. We report now the amino acid sequence of an additional human Mcm protein, p105Mcm, and show that it is homologous to the Schizosaccharomyces pombe protein Mis5. We demonstrate that the gene for protein p105Mcm (HGMW-approved symbol, MCM6) is located on human chromosome 2q14-q21. All six known human Mcm proteins have now been sequenced and compared to their yeast counterparts.

Amino Acid Sequence↗
PubMed1996

Replication protein A induces the unwinding of long double-stranded DNA regions.

We have investigated nucleoprotein filaments composed of human replication protein A (RPA) and DNA by electron microscopy. At low ionic strengths, RPA complexes with single-stranded DNA are similar in length to protein-free DNA suggesting that RPA-bound DNA remains in an extended configuration under these conditions. However, severe compaction of RPA-DNA complexes occurs in buffers with > 2 mM MgCl2 or with 100 mM NaCl. At low ionic strengths, RPA binds to A + T-rich internal regions of linear double-stranded simian virus 40 (SV40) DNA and induces separation of complementary DNA strands. RPA also binds to closed-circular SV40 DNA, but requires the function of a DNA topoisomerase to invade and completely unwind duplex DNA regions. The ability of RPA to unwind long stretches of double-stranded DNA is not shared by the bacterial single-strand binding protein and the phage T4 gene 32 protein.

Bacteriophage M13↗
PubMed1996

Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins.

Recently we identified a cDNA fragment encoding a conserved part of a new human minichromosome maintenance (Mcm) protein, provisionally termed P1.1Mcm3. Here, we report that the protein is most highly related to a yeast cell-division-cycle protein, Cdc47, encoded by the open reading frame YBR1441 on chromosome 11 of Saccharomyces cerevisiae. The human protein migrates on a polyacrylamide gel with an apparent molecular mass of 85 kDa and shares areas of significant similarity with the Mcm family of replication proteins. It is, therefore, designated as p85Mcm. Microscopic immuno-fluorescence studies revealed that protein p85Mcm is located in the nuclei of interphase cells, but is evenly distributed throughout the cell during mitosis. The amounts of p85Mcm do not significantly change during the cell cycle, but mRNA levels rise with the beginning of the S phase. However, in vitro differentiation of HL60 cells results in a striking decrease of both p85Mcm mRNA and protein levels, suggesting a role for p85Mcm in proliferating, but not in differentiated cells. Under physiological salt conditions, p85Mcm is a component of a high molecular-mass complex including other Mcm proteins. The complex dissociates at high ionic strength giving rise to stable subcomplexes, one of which contains protein p85Mcm together with Mcm proteins hCdc21 and p1O5Mcm.

Amino Acid Sequence↗
PubMed1996

Comparison of two DNA viruses infecting the marine brown algae Ectocarpus siliculosus and E. fasciculatus.

The marine brown algal genus Ectocarpus contains two species, E. siliculosus and E. fasciculatus. Field populations of both species include plants with infection symptoms caused by DNA viruses. We have established clonal cultures from infected and normal host plants and investigated the properties of the endogenous viruses. Both host species contain virus particles with a hexagonal cross-section and a diameter of ca. 150 nm. The genomes of both virus types consist of double-stranded DNA, approximately 320 kb in size. Restriction digestion with Sfil revealed differences between the two virus genomes. However, PCR experiments suggest that at least one gene, which encodes a major capsid protein, is quite similar in both virus species. In cross-infection experiments the E. siliculosus virus did not initiate an infection cycle in E. fasciculatus. In contrast, the E. fasciculatus virus infected E. siliculosus zoospores. The resulting plants showed aberrant symptoms and produced virus particles which were not infectious. We conclude that the two Ectocarpus species are hosts for different, but closely related viruses.

Capsid↗
PubMed1996

Role of amino-terminal histone domains in chromatin replication.

Simian virus 40 minichromosomes were treated with trypsin to specifically remove the amino-terminal histone domains (tails). Trypsin treatment does not affect the spacing and the number of nucleosomes on minichromosomes but indices a more extended conformation, as shown by the reduced sedimentation coefficient of trypsinized minichromosomes compared with the untreated controls. Trypsinized minichromosomes replicate more efficiently than control minichromosomes in in vitro replication assays. The increased template efficiency appears to be due to higher rates of replicative fork movement. In vitro replication in the presence of protein-free competitor DNA shows that replicating trypsinized minichromosomes do not lose nucleosomes and replicating competitor DNA does not gain nucleosomes. This finding suggests that tailless nucleosomes are transferred from the unreplicated prefork stem to replicated DNA branches and excludes a participation of the basic histone domains in nucleosome transfer.

Animals↗
PubMed1996

Coding sequence and chromosome mapping of the human gene (CDC46) for replication protein hCdc46/Mcm5.

We have isolated the complete cDNA sequence encoding the human homolog of the yeast replication protein Cdc46/Mcm5. The cDNA was used as a probe for chromosomal fluorescence in situ hybridization (FISH), which localized the human Cdc46 gene (CDC46) to chromosome region 22q13.1-->q13.2. This is the fourth human Mcm gene whose chromosome region has been determined, and it is now apparent that human Mcm genes are widely distributed in the genome. As deduced from the cDNA sequence, human protein hCdc46 is composed of 734 amino acids and contains a central region that is almost 80% identical with the yeast protein. Immunoprecipitation experiments with hCdc46-specific antibodies confirm that essentially all nuclear hCdc46 proteins form a stable dimeric complex with protein P1Mcm3.

Amino Acid Sequence↗
PubMed1996

Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication.

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.

Animals↗
PubMed1995

A human homologue of the yeast replication protein Cdc21. Interactions with other Mcm proteins.

We present the amino acid sequence of the human homologue of the yeast replication protein Cdc21, a member of the Mcm family of nuclear proteins. Specific antibodies, raised against protein hCdc21, were used to investigate the expression of the protein through the cell cycle. The protein is highly phosphorylated in mitotic cells. The phosphorylated form of protein hCdc21 appears to be less tightly bound to nuclear structures than the underphosphorylated form suggesting that phosphorylation/dephosphorylation reactions may determine the nuclear distribution of the protein. Protein hCdc21 forms a stable trimeric complex with two novel human Mcm proteins, p85Mcm and p105Mcm. Protein BM28/Mcm2 is more loosely associated with the trimeric hCdc21 complex.

Amino Acid Sequence↗
PubMed1995

Interactions of human nuclear proteins P1Mcm3 and P1Cdc46.

Human nuclear proteins P1Mcm3 and P1Cdc46 have high sequence similarities with the corresponding yeast proteins known to be required for the initiation of genome replication. Nuclei of proliferating HeLa cells contain relatively high amounts of P1Mcm3 (about 10(6) molecules/nucleus) of which only a small fraction is bound to a nuclear structure, most probably chromatin. At 0.5 M NaCl, the structure-bound nuclear protein can be partially solubilized as a dimer composed of P1Mcm3 and the related protein P1Cdc46. However, most protein P1Mcm3 is not bound to a nuclear structure and appears in the nucleoplasm. About 10% of protein P1Mcm3 in the soluble fraction is free and uncomplexed, and the remaining P1Mcm3 forms stable complexes with protein P1Cdc46. These P1Mcm3/Cdc46 complexes occur as dimers and in high-molecular-mass complexes (approximately 500 kDa). The high-molecular-mass complexes dissociate in 0.5 M NaCl and release P1Mcm3/Cdc46 dimers. It has frequently been proposed that the Mcm proteins may function as licensing factors for genome replication. Our data imply that the active form of an Mcm protein is not a monomer, but a protein complex that includes an Mcm3/Cdc46 dimer. DNA polymerase alpha is not a component of this complex.

Amino Acid Sequence↗
PubMed1995

Coat protein of the Ectocarpus siliculosus virus.

Ectocarpus siliculosus virus, EsV, multiplies in sporangia and gametangia of the marine brown alga Ectocarpus siliculosus. We describe an improved method for the isolation of morphologically intact and infectious virus from diseased plants. We show that treatment of virus particles with high concentrations of CsCl results in a substantial loss of structural proteins. One of the proteins which resists CsCl treatment is glycoprotein-1, the largest of the three viral glycoproteins. We have isolated an EsV genomic fragment with an open reading frame encoding glycoprotein-1. The predicted amino acid sequence is rich in hydrophilic amino acids, but contains hydrophobic regions close to the amino and carboxy termini. A discrepancy between the molecular weight predicted from the coding region and the molecular weight determined by gel electrophoresis suggests that proteolytic processing is required for the maturation of the protein.

Amino Acid Sequence↗