EMBO Conference on Replication and Segregation of Chromosomes, Geilo, Norway, June 16-20. Replication and segregation of chromosomes in the three domains of life: EMBO conference reports common grounds. Meeting report.
Journal - Plasmid (United States )
A meeting of the EMBO Conference Series on Replication and Segregation of Chromosomes was held in Geilo, Norway, 16-20 June, 2008, under a scenic backdrop of high mountains. The meeting focused on the mechanistic details of replication and segregation primarily from well-characterized systems. Because the same basic principles govern chromosome maintenance in all three domains of life, participants encountering parallel processes in distantly-related organisms were stimulated to interact. Another successful aspect of the meeting was the quality of the posters, several of which were chosen for platform presentation and two for special rewards. The organizers Kirsten Skarstad and Erik Boye deserve praise for their skillful organization of the meeting, the highlights of which are discussed below.
|ISSN : ||1095-9890|
|Mesh Heading : ||Animals Bacteria Cell Division Chromosome Segregation Chromosomes DNA Replication Timing Humans Norway Plasmids genetics genetics genetics genetics genetics genetics physiology|
|Mesh Heading Relevant : ||physiology physiology physiology physiology|
Replication in context: dynamic regulation of DNA replication patterns in metazoans.
Journal - Nature reviews. Genetics (England )
Replication in eukaryotes initiates from discrete genomic regions according to a strict, often tissue-specific temporal programme. However, the locations of initiation events within initiation regions vary, show sequence disparity and are affected by interactions with distal elements. Increasing evidence suggests that specification of replication sites and the timing of replication are dynamic processes that are regulated by tissue-specific and developmental cues, and are responsive to epigenetic modifications. Dynamic specification of replication patterns might serve to prevent or resolve possible spatial and/or temporal conflicts between replication, transcription and chromatin assembly, and facilitate subtle or extensive changes of gene expression during differentiation and development.
|ISSN : ||1471-0056|
|Mesh Heading : ||Animals Binding Sites Cell Differentiation DNA Replication Embryonic Development Genomic Instability Humans Models, Genetic Multiprotein Complexes Origin Recognition Complex Transcription, Genetic chemistry metabolism|
|Mesh Heading Relevant : ||genetics physiology|
Molecular interaction maps--a diagrammatic graphical language for bioregulatory networks.
Journal - Science's STKE : signal transduction knowledge environment (United States )
Molecular interaction maps (MIMs) use a clear, accurate, and versatile graphical language to depict complex biological processes. Here, we discuss the main features of the MIM language and its potential uses. MIMs can be used as database resources and simulation guides, and can serve to generate new hypotheses regarding the roles of specific molecules in the bioregulatory networks that control progression through the cell cycle, differentiation, and cell death.
|ISSN : ||1525-8882|
|Mesh Heading : ||Animals Computational Biology Gene Expression Regulation Protein Interaction Mapping Signal Transduction|
|Mesh Heading Relevant : ||Computer Graphics Databases, Protein methods statistics & numerical data genetics methods genetics|
The replicon revisited: an old model learns new tricks in metazoan chromosomes.
Journal - EMBO reports (England )
The origins of DNA replication were proposed in the replicon model to be specified genetically by replicator elements that coordinate the initiation of DNA synthesis with gene expression and cell growth. Recent studies have identified DNA sequences in mammalian cells that fulfil the genetic criteria for replicators and are beginning to uncover the sequence requirements for the initiation of DNA replication. Mammalian replicators are com- posed of non-redundant modules that cooperate to direct initiation to specific chromosomal sites. Conversely, replicators do not show strong sequence similarity, and their ability to initiate replication depends on the chromosomal context and epigenetic factors, as well as their primary sequence. Here, we review the properties of metazoan replicators, and discuss the genetic and epigenetic factors that determine where and when DNA replication is initiated.
|ISSN : ||1469-221X|
|Mesh Heading : ||Animals Cell Proliferation Chromatin Chromosomes CpG Islands DNA DNA Methylation DNA Replication Humans Models, Genetic Protein Structure, Tertiary Transcription, Genetic chemistry metabolism metabolism|
|Mesh Heading Relevant : ||Replication Origin ultrastructure ultrastructure|
The mammalian beta globin origin of DNA replication.
Journal - Frontiers in bioscience : a journal and virtual library (United States )
Initiation of DNA replication is a tightly regulated process aimed to insure that the entire genome is replicated at the appropriate time during each cell cycle. In the human beta globin locus, replication initiates from a region between the two genes that encode the adult subunit of hemoglobin (the beta globin initiation region, or IR). Mammalian beta globin loci replicate early during the S phase of the cell cycle in pre erythroid cells, in which the beta-globin locus is present in a euchromatin form. However, in cells that do not express globin and in which the locus is heterochromatic, these same loci replicate during the later stages of S phase. Both early and late replication patterns utilize similar replication initiation regions. These features make the beta globin locus an attractive model for studying the determinants of replication sites and replication timing, as well as the correlation between gene expression and DNA replication. Two genomic domains are essential for initiation of DNA replication within the locus: the initiation region (IR), and a 40 kb region upstream of the globin gene cluster known as the locus control region (LCR). The IR meets the genetic requirements for a chromosomal replicator, since it can initiate DNA replication at ectopic sites. The LCR regulates transcriptional activity and chromatin structure, and may act as a determinant of replication timing. This review will summarize recent findings characterizing the sequence requirements for initiation of DNA replication in mammalian beta globin loci and will discuss the specific influence of the location and the chromosomal environment in regulating DNA replication at the beta globin IR.
|ISSN : ||1093-4715|
|Mesh Heading : ||Animals Binding Sites Cell Cycle Chromatin Chromosomes DNA DNA Replication Euchromatin Globins Heterochromatin Humans Multigene Family Time Factors Transcription, Genetic metabolism metabolism chemistry metabolism metabolism metabolism|
|Mesh Heading Relevant : ||Gene Expression Regulation Replication Origin genetics|
Chromatin Challenges during DNA Replication: A Systems Representation
Journal - Molecular Biology of the Cell
In a recent review, A. Groth and coworkers presented a comprehensive account of nucleosome disassembly in front of a DNA replication fork, assembly behind the replication fork, and the copying of epigenetic information onto the replicated chromatin. Understanding those processes however would be enhanced by a comprehensive graphical depiction analogous to a circuit diagram. Accordingly, we have constructed a molecular interaction map (MIM) that preserves in essentially complete detail the processes described by Groth et al. The MIM organizes and elucidates the information presented by Groth et al. on the complexities of chromatin replication, thereby providing a tool for system-level comprehension of the effects of genetic mutations, altered gene expression, and pharmacologic intervention.
The Intra-S-Phase Checkpoint Affects both DNA Replication Initiation and Elongation: Single-Cell and -DNA Fiber Analyses
Journal - Molecular and Cellular Biology
To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone ?-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.
The Human ß-Globin Locus Control Region Can Silence as Well as Activate Gene Expression
Journal - Molecular and Cellular Biology
Using recombinase-mediated cassette exchange to test multiple transgenes at the same site of integration, we demonstrate a novel chromatin context-dependent silencer activity of the ß-globin locus control region (LCR). This silencer activity requires DNase I hypersensitive sites HS2 and HS3 but not HS4. After silencing, the silenced cassettes adopt a typical closed chromatin conformation (histone H3 and H4 deacetylation, histone H3-K4 methylation, DNA methylation, and replication in late S phase). In the absence of the LCR at the same site of integration, the chromatin remains decondensed. We demonstrate that the LCR is necessary but not sufficient to trigger these chromatin changes. We also provide evidence that this novel silencing activity is caused by transcriptional interference triggered by activation of transcription in the flanking sequences by the LCR.
The Human ß-Globin Replication Initiation Region Consists of Two Modular Independent Replicators
Journal - Molecular and Cellular Biology
Previous studies have shown that mammalian cells contain replicator sequences, which can determine where DNA replication initiates. However, the specific sequences that confer replicator activity were not identified. Here we report a detailed analysis of replicator sequences that dictate initiation of DNA replication from the human ß-globin locus. This analysis suggests that the ß-globin replication initiation region contains two adjacent, redundant replicators. Each replicator was capable of initiating DNA replication independently at ectopic sites. Within each of these two replicators, we identified short, discrete, nonredundant sequences, which cooperatively determine replicator activity. Experiments with somatic cell hybrids further demonstrated that the requirements for initiation at ectopic sites were similar to the requirements for initiation within native human chromosomes. The replicator clustering and redundancy exemplified in the human ß-globin locus may account for the extreme difficulty in identifying replicator sequences in mammalian cells and suggest that mammalian replication initiation sites may be determined by cooperative sequence modules.
Replication Initiation Patterns in the ß-Globin Loci of Totipotent and Differentiated Murine Cells: Evidence for Multiple Initiation Regions
Journal - Molecular and Cellular Biology
The replication initiation pattern of the murine ß-globin locus was analyzed in totipotent embryonic stem cells and in differentiated cell lines. Initiation events in the murine ß-globin locus were detected in a region extending from the embryonic Ey gene to the adult ßminor gene, unlike the restricted initiation observed in the human locus. Totipotent and differentiated cells exhibited similar initiation patterns. Deletion of the region between the adult globin genes did not prevent initiation in the remainder of the locus, suggesting that the potential to initiate DNA replication was not contained exclusively within the primary sequence of the deleted region. In addition, a deletion encompassing the six identified 5' hypersensitive sites in the mouse locus control region had no effect on initiation from within the locus. As this deletion also did not affect the chromatin structure of the locus, we propose that the sequences determining both chromatin structure and replication initiation lie outside the hypersensitive sites removed by the deletion.
Initiation of DNA replication at the human ß-globin 3' enhancer
Journal - Nucleic Acids Research
The origin of DNA replication in the human ß-globingene contains an initiation region (IR) and two flanking auxiliaryelements. Two replicator modules are located within the upstreamauxiliary sequence and the IR core, but the functional sequencesin the downstream auxiliary element are unknown. Here, we usea combination of benzoylated-naphthoylated DEAE (BND) cellulosepurification and nascent strand abundance assays to show thatreplication initiation occurs at the ß-globin 3' enhanceron human chromosome 11 in the Hu11 hybrid murine erythroleukemia(MEL) cell line. To examine replicator function, 3' enhancerfragments were inserted into an ectopic site in MEL cells viaan optimized FRT/EGFP-FLP integration system. These experimentsdemonstrate that the 1.6 kb downstream auxiliary element isa third replicator module called bGRep-E in erythroid cells.The minimal 260 bp 3' enhancer is required but not sufficientto initiate efficient replication, suggesting cooperation withadjacent sequences. The minimal 3' enhancer also cooperateswith elements in an expressing HS3ß/-globin constructto initiate replication. These data indicate that the ß-globinreplicator has multiple initiation sites in three closely spacedreplicator modules. We conclude that a mammalian enhancer cancooperate with adjacent sequences to create an efficient replicatormodule.
Targeted Deletion of the Chicken -Globin Regulatory Elements Reveals a Cooperative Gene Silencing Activity*
Journal - Journal of Biological Chemistry
The chicken -globin locus represents a well characterized systemto study the role of both proximal and distal regulatory elementsin a eukaryotic multigene domain. The function of the chickenA/-intergenic enhancer and upstream regulatory elements 5'-HS1and 5'-HS2 were studied using a gene targeting approach in chickenDT40 cells followed by microcell-mediated chromosome transferinto human erythroleukemia cells (K562). These regulatory elementsall repressed expression of the - and H-chicken globin genesin the chromosome transfer assay. No - or H-globin gene expressionwas detected in K562 cells containing the chicken chromosomewithout deletions, whereas - and H-mRNA was activated in K562cells containing chicken chromosomes with deletions of the intergenicenhancers, 5'-HS1 and 5'-HS2. Transcriptional activation ofthe - and H-globin genes correlated with hyperacetylation ofhistones H3 and H4, loss of histone H3 lysine 9 methylation,and binding of RNA polymerase II to the gene promoters. Surprisingly,the status of CpG dinucleotide methylation at the promotersdid not correlate with the transcriptional status of the genes.Our results using a chromosomal transfer assay demonstrate anidentical silencing function for these regulatory elements,which suggests they function as part of a common silencing pathwayor complex.* This work was supported by National Institutes of Health GrantDK56798. The costs of publication of this article were defrayedin part by the payment of page charges. This article must thereforebe hereby marked "advertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact. The on-line version of this article (available at http://www.jbc.org)contains supplemental Tables I and II. Present address: Virginia Bioinformatics Institute, Blacksburg,Virginia 24061.
Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes*
Journal - Journal of Biological Chemistry
DNA double-strand breaks originating from diverse causes ineukaryotic cells are accompanied by the formation of phosphorylatedH2AX (H2AX) foci. Here we show that H2AX formation is alsoa cellular response to topoisomerase I cleavage complexes knownto induce DNA double-strand breaks during replication. In HCT116human carcinoma cells exposed to the topoisomerase I inhibitorcamptothecin, the resulting H2AX formation can be preventedwith the phosphatidylinositol 3-OH kinase-related kinase inhibitorwortmannin; however, in contrast to ionizing radiation, only camptothecin-induced H2AX formation can be prevented with theDNA replication inhibitor aphidicolin and enhanced with thecheckpoint abrogator 7-hydroxystaurosporine. This H2AX formationis suppressed in ATR (ataxia telangiectasia and Rad3-related)deficient cells and markedly decreased in DNA-dependent proteinkinase-deficient cells but is not abrogated in ataxia telangiectasiacells, indicating that ATR and DNA-dependent protein kinaseare the kinases primarily involved in H2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11-and Nbs1-deficient cells are still able to form H2AX. However,H2AX-/- mouse embryonic fibroblasts exposed to camptothecinfail to form Mre11, Rad50, and Nbs1 foci and are hypersensitiveto camptothecin. These results demonstrate a conserved H2AXresponse for double-strand breaks induced by replication forkcollision. H2AX foci are required for recruiting repair andcheckpoint protein complexes to the replication break sites.* The costs of publication of this article were defrayed in partby the payment of page charges. This article must thereforebe hereby marked "advertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.