Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner.
TL;DR: It is shown that 0.5 mM mimosine can induce a cell cycle arrest of human somatic cells in late G1 phase, before establishment of active DNA replication forks, which can be exploited for studying the initiation of human DNA replication in vitro.
About: This article is published in Experimental Cell Research.The article was published on 1999-02-25. It has received 175 citations till now. The article focuses on the topics: Mimosine & DNA replication.
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TL;DR: A stepwise model for the formation of a transcriptionally silent heterochromatin is provided: SUV39H1 places a ‘methyl marker’ on histone H3, which is then recognized by HP1 through its chromo domain, which may also explain the stable inheritance of theheterochromatic state.
Abstract: Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homologue of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 methylate specifically lysine 9 of histone H3 (ref. 6). Here we show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state.
2,811 citations
Cites methods from "Mimosine arrests proliferating huma..."
...Preparation of nuclei and peptide challenge U2OS nuclei were purifie...
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TL;DR: DNMT1 not only maintains DNA methylation, but also may directly target, in a heritable manner, transcriptionally repressive chromatin to the genome during DNA replication.
Abstract: DNA methylation can contribute to transcriptional silencing through several transcriptionally repressive complexes, which include methyl-CpG binding domain proteins (MBDs) and histone deacetylases (HDACs) We show here that the chief enzyme that maintains mammalian DNA methylation, DNMT1, can also establish a repressive transcription complex The non-catalytic amino terminus of DNMT1 binds to HDAC2 and a new protein, DMAP1 (for DNMT1 associated protein), and can mediate transcriptional repression DMAP1 has intrinsic transcription repressive activity, and binds to the transcriptional co-repressor TSG101 DMAP1 is targeted to replication foci through interaction with the far N terminus of DNMT1 throughout S phase, whereas HDAC2 joins DNMT1 and DMAP1 only during late S phase, providing a platform for how histones may become deacetylated in heterochromatin following replication Thus, DNMT1 not only maintains DNA methylation, but also may directly target, in a heritable manner, transcriptionally repressive chromatin to the genome during DNA replication
1,062 citations
TL;DR: Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1α at the same site.
Abstract: Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. Here we show that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1alpha (HP1alpha), but not HP1beta, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1alpha at the same site. Tauhese results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia.
578 citations
Additional excerpts
...Anti-rabbit IgG H3(31-56) peptide pulldown...
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TL;DR: The data indicate that the monoubiquitination of FANCD2 is highly regulated, and they suggest that FANcd2/BRCA1 complexes and FAN CD2/RAD51 complexes participate in an S-phase-specific cellular process, such as DNA repair by homologous recombination.
472 citations
TL;DR: The results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB by creating SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions.
Abstract: DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.
346 citations
Cites background from "Mimosine arrests proliferating huma..."
...human cells (20): cells were incubated in medium containing 0....
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References
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TL;DR: In this article, it was shown that the enzymes might have evolved from a common ancestor, with the class III anaerobic Escherichia coli reductase as its closest relative.
Abstract: It is generally accepted that DNA appeared after RNA during the chemical evolution of life. To synthesize DNA, deoxyribonucleotides are required as building blocks. At present, these are formed from the corresponding ribonucleotides through the enzymatic action of ribonucleotide reductases. Three classes of enzymes are present in various organisms. There is little sequence similarity among the three classes of reductases. However, enzymic mechanisms and the allosteric behavior of the enzymes from various organisms are strongly conserved, suggesting that the enzymes might have evolved from a common ancestor, with the class III anaerobic Escherichia coli reductase as its closest relative.
523 citations
TL;DR: HeLa cells in early S phase were encapsulated in agarose microbeads, permeabilized, and incubated with biotin-11-dUTP in a "physiological" buffer to provide visual evidence for polymerization "factories" fixed to a skeleton, with replication occurring as the template moves through them.
462 citations
420 citations
TL;DR: A cell-free system from HeLa cells that initiates DNA replication under cell cycle control is described that could be replaced by recombinant human cyclins A and E complexed to C DK2 but not by Cdk2 alone or by human cyclin B1 complexing to Cdc2.
319 citations
TL;DR: Nuclei prepared by the above method look morphologically healthy in oocytes cultured in vitro for up to one month after nuclear injection, compared with other methods, such as those involving the use of detergents, which undergo deterioration within a few days after injection into oocytes.
Abstract: A method is described by which nuclei associated with some cytoplasm can be rapidly prepared from a suspension of cells. The method involves the use of lysolecithin and bovine serum albumin. Oocytes of Xenopus laevis were injected with about 200 nuclei prepared from human He La cells by this method. Nuclei were deposited in oocyte cytoplasm, in the oocyte nucleus, or in the dispersed contents of a ruptured oocyte nucleus. Injected He La nuclei enlarge up to several hundred times in volume in the course of a few days. Their enlargement is associated with chromatin dispersion, increased binding of an acidic dye, and with the reduction in size, and eventual disappearance, of nucleoli. The amount of He La nucleus enlargement is much greater when the oocyte nucleus is ruptured. The fate of injected nuclei was followed by the use of HeLa nuclei whose DNA had been previously la belled with [ 3 H] thymidine. La belled DNA does not pass from injected He La nuclei into the oocyte nucleus. Injected nuclei appear not to fuse with each other or with the oocyte nucleus. Nuclei prepared by the above method look morphologically healthy in oocytes cultured in vitro for up to one month after nuclear injection. Nuclei prepared by other methods, such as those involving the use of detergents, undergo deterioration within a few days after injection into oocytes.
290 citations