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1
Marini, N. J., and R. M. Benbow. "Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency." Molecular and Cellular Biology 11, no. 1 (January 1991): 299–308. http://dx.doi.org/10.1128/mcb.11.1.299.
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Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).
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Marini, N. J., and R. M. Benbow. "Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency." Molecular and Cellular Biology 11, no. 1 (January 1991): 299–308. http://dx.doi.org/10.1128/mcb.11.1.299-308.1991.
Full textAbstract:
Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).
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Franks, R. R., B. R. Hough-Evans, R. J. Britten, and E. H. Davidson. "Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA." Development 102, no. 2 (February 1, 1988): 287–99. http://dx.doi.org/10.1242/dev.102.2.287.
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A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.
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Meng, Yuan, Changwei Liu, Lei Shen, Mian Zhou, Wenpeng Liu, Claudia Kowolik, Judith L. Campbell, Li Zheng, and Binghui Shen. "TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity." Nucleic Acids Research 47, no. 14 (June 19, 2019): 7564–79. http://dx.doi.org/10.1093/nar/gkz537.
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Abstract The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.
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Liang, Li, Li Deng, Yanping Chen, Gloria C. Li, Changshun Shao, and Jay A. Tischfield. "Modulation of DNA End Joining by Nuclear Proteins." Journal of Biological Chemistry 280, no. 36 (July 11, 2005): 31442–49. http://dx.doi.org/10.1074/jbc.m503776200.
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Irianto, Jerome, Charlotte R. Pfeifer, Rachel R. Bennett, Yuntao Xia, Irena L. Ivanovska, Andrea J. Liu, Roger A. Greenberg, and Dennis E. Discher. "Nuclear constriction segregates mobile nuclear proteins away from chromatin." Molecular Biology of the Cell 27, no. 25 (December 15, 2016): 4011–20. http://dx.doi.org/10.1091/mbc.e16-06-0428.
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As a cell squeezes its nucleus through adjacent tissue, penetrates a basement membrane, or enters a small blood capillary, chromatin density and nuclear factors could in principle be physically perturbed. Here, in cancer cell migration through rigid micropores and in passive pulling into micropipettes, local compaction of chromatin is observed coincident with depletion of mobile factors. Heterochromatin/euchromatin was previously estimated from molecular mobility measurements to occupy a volume fraction f of roughly two-thirds of the nuclear volume, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to nearly 100%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g., BRCA1, 53BP1) or nucleases (e.g., Cas9, FokI), are depleted within the constriction, approaching 0%. Such losses—compounded by the occasional rupture of the nuclear envelope—can have important functional consequences. Studies of a nuclease that targets a locus in chromosome-1 indeed show that constricted migration delays DNA damage.
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Ma, Hong, Alan J. Siegel, and Ronald Berezney. "Association of Chromosome Territories with the Nuclear Matrix." Journal of Cell Biology 146, no. 3 (August 9, 1999): 531–42. http://dx.doi.org/10.1083/jcb.146.3.531.
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To study the possible role of the nuclear matrix in chromosome territory organization, normal human fibroblast cells are treated in situ via classic isolation procedures for nuclear matrix in the absence of nuclease (e.g., DNase I) digestion, followed by chromosome painting. We report for the first time that chromosome territories are maintained intact on the nuclear matrix. In contrast, complete extraction of the internal nuclear matrix components with RNase treatment followed by 2 M NaCl results in the disruption of higher order chromosome territory architecture. Correlative with territorial disruption is the formation of a faint DNA halo surrounding the nuclear lamina and a dispersive effect on the characteristically discrete DNA replication sites in the nuclear interior. Identical results were obtained using eight different human chromosome paints. Based on these findings, we developed a fractionation strategy to release the bulk of nuclear matrix proteins under conditions where the chromosome territories are maintained intact. A second treatment results in disruption of the chromosome territories in conjunction with the release of a small subset of acidic proteins. These proteins are distinct from the major nuclear matrix proteins and may be involved in mediating chromosome territory organization.
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Meier, J., K. H. Campbell, C. C. Ford, R. Stick, and C. J. Hutchison. "The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs." Journal of Cell Science 98, no. 3 (March 1, 1991): 271–79. http://dx.doi.org/10.1242/jcs.98.3.271.
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Xenopus egg extracts, which support nuclear assembly and DNA replication, were functionally depleted of lamin LIII by inoculating them with monoclonal anti-lamin antibodies. Phase-contrast microscopy and electron-microscopy studies indicated that lamin-depleted extracts supported efficient chromatin decondensation, and assembly of double membrane structures and nuclear pores on demembranated sperm heads. Immunofluorescence microscopy suggests that lamin-antibody complexes are transported across the nuclear membrane but do not assemble into a lamina. These findings were confirmed by immunoblotting analysis of isolated nuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking a lamina were unable to initiate DNA replication and that, although such nuclei could import proteins required for DNA replication (e.g. PCNA), these proteins were apparently not organized into replicon clusters.
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Büsse, Sebastian, Philipp von Grumbkow, Janine Mazanec, Gert Tröster, Susanne Hummel, and Thomas Hörnschemeyer. "Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA." Entomological Science 20, no. 1 (January 2017): 137–41. http://dx.doi.org/10.1111/ens.12242.
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Ceppi, Ilaria, Sean M. Howard, Kristina Kasaciunaite, Cosimo Pinto, Roopesh Anand, Ralf Seidel, and Petr Cejka. "CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection." Proceedings of the National Academy of Sciences 117, no. 16 (April 2, 2020): 8859–69. http://dx.doi.org/10.1073/pnas.2001165117.
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To repair a DNA double-strand break by homologous recombination, 5′-terminated DNA strands must first be resected to reveal 3′-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5′-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5′-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.
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Deville, Sara Sofia, Anne Vehlow, Sarah Förster, Ellen Dickreuter, Kerstin Borgmann, and Nils Cordes. "The Intermediate Filament Synemin Regulates Non-Homologous End Joining in an ATM-Dependent Manner." Cancers 12, no. 7 (June 28, 2020): 1717. http://dx.doi.org/10.3390/cancers12071717.
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The treatment resistance of cancer cells is a multifaceted process in which DNA repair emerged as a potential therapeutic target. DNA repair is predominantly conducted by nuclear events; yet, how extra-nuclear cues impact the DNA damage response is largely unknown. Here, using a high-throughput RNAi-based screen in three-dimensionally-grown cell cultures of head and neck squamous cell carcinoma (HNSCC), we identified novel focal adhesion proteins controlling DNA repair, including the intermediate filament protein, synemin. We demonstrate that synemin critically regulates the DNA damage response by non-homologous end joining repair. Mechanistically, synemin forms a protein complex with DNA-PKcs through its C-terminal tail domain for determining DNA repair processes upstream of this enzyme in an ATM-dependent manner. Our study discovers a critical function of the intermediate filament protein, synemin in the DNA damage response, fundamentally supporting the concept of cytoarchitectural elements as co-regulators of nuclear events.
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Xue, Chaoyou, Weibin Wang, J. Brooks Crickard, Corentin J. Moevus, Youngho Kwon, Patrick Sung, and Eric C. Greene. "Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection." Proceedings of the National Academy of Sciences 116, no. 13 (March 8, 2019): 6091–100. http://dx.doi.org/10.1073/pnas.1819276116.
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In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3′ single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1–dependent or –independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1–Dna2–Top3-Rmi1–RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.
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Lu, Z. H., D. B. Sittman, D. T. Brown, R. Munshi, and G. H. Leno. "Histone H1 modulates DNA replication through multiple pathways in Xenopus egg extract." Journal of Cell Science 110, no. 21 (November 1, 1997): 2745–58. http://dx.doi.org/10.1242/jcs.110.21.2745.
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We investigated the effects of histone H1s on DNA replication using Xenopus egg extract. Mouse variants H1c and H10 were assembled onto Xenopus sperm chromatin by the extract during the remodeling that accompanies nuclear decondensation. The association of H1 with chromatin was rapid and concentration dependent. H1-associated chromatin displayed a typical nucleosomal repeat pattern indicating that linker histones are properly positioned along the DNA. The presence of H1 on sperm chromatin reduced both the rate and extent of DNA replication in egg extract. This reduction in rate is due, in part, to a delay in initiation of replication within individual nuclei. Initiation in extract is dependent upon nuclear assembly. Analysis of the assembly process revealed that H1 does not inhibit nuclear membrane formation or the import of nuclear protein, however, it does slow the rate of nuclear lamina formation. This H1-induced delay in lamina assembly is responsible for the delay in initiation as pre-assembled H1-containing nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete suggesting that H1 also affects replication directly. These data indicate that H1 modulates DNA replication through multiple pathways in egg extract.
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Trempe, J. P., Y. I. Lindstrom, and M. Leffak. "Opposite replication polarities of transcribed and nontranscribed histone H5 genes." Molecular and Cellular Biology 8, no. 4 (April 1988): 1657–63. http://dx.doi.org/10.1128/mcb.8.4.1657.
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We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.
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Trempe, J. P., Y. I. Lindstrom, and M. Leffak. "Opposite replication polarities of transcribed and nontranscribed histone H5 genes." Molecular and Cellular Biology 8, no. 4 (April 1988): 1657–63. http://dx.doi.org/10.1128/mcb.8.4.1657-1663.1988.
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We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.
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Gilbert, D. M., H. Miyazawa, and M. L. DePamphilis. "Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure." Molecular and Cellular Biology 15, no. 6 (June 1995): 2942–54. http://dx.doi.org/10.1128/mcb.15.6.2942.
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Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.
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Pittoggi, C., L. Renzi, G. Zaccagnini, D. Cimini, F. Degrassi, R. Giordano, A. R. Magnano, R. Lorenzini, P. Lavia, and C. Spadafora. "A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA." Journal of Cell Science 112, no. 20 (October 15, 1999): 3537–48. http://dx.doi.org/10.1242/jcs.112.20.3537.
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We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of ‘active’ chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.
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Rothbarth, K., E. Spiess, B. Juodka, U. Yavuzer, P. Nehls, H. Stammer, and D. Werner. "Induction of apoptosis by overexpression of the DNA-binding and DNA-PK-activating protein C1D." Journal of Cell Science 112, no. 13 (July 1, 1999): 2223–32. http://dx.doi.org/10.1242/jcs.112.13.2223.
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Apoptosis is induced in various tumor cell lines by vector-dependent overexpression of the conserved gene C1D that encodes a DNA-binding and DNA-PK-activating protein. C1D is physiologically expressed in 50 human tissues tested, which points to its basic cellular function. The expression of this gene must be tightly regulated because elevated levels of C1D protein, e.g. those induced by transient vector-dependent expression, result in apoptotic cell death. Cells transfected with C1D-expressing constructs show terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of DNA ends. Transfections with constructs in which C1D is expressed in fusion with the (enhanced) green fluorescent protein from A. victoria (EGFP) allow the transfected cells to be identified and the morphological changes induced to be traced. Starting from intense nuclear spots, green fluorescence reflecting C1D expression increases dramatically at 12–24 hours post-transfection. Expression of C1D-EGFP protein is accompanied by morphological changes typical of apoptotic cell death, e.g. cytoplasmic vacuolation, membrane blebbing and nuclear disintegration. Cell shrinkage and detachment from extracellular matrix are observed in monolayer cultures while suspension cells become progressively flattened. The facility to differentiate between transfected and non-transfected cells reveals that non-transfected cells co-cultured with transfected cells also show the morphological changes of apoptosis, which points to a bystander effect. C1D-dependent apoptosis is not induced in cells with non-functional p53. Accordingly, C1D-induced apoptosis is discussed in relation to its potential to activate DNA-PK, which has been considered to act as an upstream activator of p53.
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Blow, J. J., and A. M. Sleeman. "Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly." Journal of Cell Science 95, no. 3 (March 1, 1990): 383–91. http://dx.doi.org/10.1242/jcs.95.3.383.
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Purified DNA undergoes a single round of semiconservative replication when incubated in extracts of Xenopus eggs. These extracts also assemble purified DNA into pseudo-nuclei, structures closely resembling normal interphase nuclei. In this paper we show that although less than 60% of purified DNA is assembled into pseudo-nuclei, DNA replication takes place only within these pseudo-nuclei. Further, when nuclear assembly is prevented, the initiation of replication on purified DNA molecules does not occur. In contrast to previous reports, we show that the initiation of DNA replication occurs only during interphase and not during mitosis, even when very high concentrations of purified DNA are used. These experiments show that nuclear formation is a general requirement for the initiation of DNA replication in this system.
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Hola, M., S. Castleden, M. Howard, and R. F. Brooks. "Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts." Journal of Cell Science 107, no. 11 (November 1, 1994): 3045–53. http://dx.doi.org/10.1242/jcs.107.11.3045.
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Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.
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Heijo, Hiroko, Sora Shimogama, Shuichi Nakano, Anna Miyata, Yasuhiro Iwao, and Yuki Hara. "DNA content contributes to nuclear size control in Xenopus laevis." Molecular Biology of the Cell 31, no. 24 (November 15, 2020): 2703–17. http://dx.doi.org/10.1091/mbc.e20-02-0113.
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Lawrence, Katherine S., Erin C. Tapley, Victor E. Cruz, Qianyan Li, Kayla Aung, Kevin C. Hart, Thomas U. Schwartz, Daniel A. Starr, and JoAnne Engebrecht. "LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining." Journal of Cell Biology 215, no. 6 (December 12, 2016): 801–21. http://dx.doi.org/10.1083/jcb.201604112.
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The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.
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Maggio, Ignazio, Hidde A. Zittersteijn, Qian Wang, Jin Liu, Josephine M. Janssen, Ivan Toral Ojeda, Silvère M. van der Maarel, Arjan C. Lankester, Rob C. Hoeben, and Manuel A. F. V. Gonçalves. "Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components." Gene Therapy 27, no. 5 (January 3, 2020): 209–25. http://dx.doi.org/10.1038/s41434-019-0119-y.
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AbstractEnhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.
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Stokes, Matthew P., and W. Matthew Michael. "DNA damage-induced replication arrest in Xenopus egg extracts." Journal of Cell Biology 163, no. 2 (October 27, 2003): 245–55. http://dx.doi.org/10.1083/jcb.200306006.
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Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase α, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase α and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.
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Smilenov, Lubomir B., Sonu Dhar, and Tej K. Pandita. "Altered Telomere Nuclear Matrix Interactions and Nucleosomal Periodicity in Ataxia Telangiectasia Cells before and after Ionizing Radiation Treatment." Molecular and Cellular Biology 19, no. 10 (October 1, 1999): 6963–71. http://dx.doi.org/10.1128/mcb.19.10.6963.
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ABSTRACT Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G1, G2, and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATMgene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene.
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Murphy, J., C. M. Crompton, S. Hainey, G. A. Codd, and C. J. Hutchison. "The role of protein phosphorylation in the assembly of a replication competent nucleus: investigations in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR." Journal of Cell Science 108, no. 1 (January 1, 1995): 235–44. http://dx.doi.org/10.1242/jcs.108.1.235.
Full textAbstract:
Cell-free extracts of Xenopus eggs support nuclear assembly and DNA replication in vitro. Extracts supplemented with the protein phosphatase inhibitor microcystin-LR displayed various inhibitory effects at different concentrations of the toxin. In the presence of cycloheximide, additions of microcystin did not induce histone H1-kinase activity. Nevertheless, increasing concentrations of microcystin did sequentially prevent DNA replication, nuclear lamina assembly and nuclear envelope assembly. DNA replication was prevented when microcystin was added at 250 nM. Furthermore, this effect could be reversed after the addition of the catalytic sub-unit of protein phosphatase 2A to inhibited extracts. At a concentration of 250 nM microcystin, nuclear membrane assembly, nuclear lamina assembly and nuclear transport all occurred in egg extracts. In addition single-stranded M13 DNA replication was also permitted. However, it appeared that replicase assembly was not completed, since nuclei assembled in microcystin-treated extracts displayed an unusual distribution of proliferating cell nuclear antigen (PCNA). Although PCNA was located at sites that resembled pre-replication foci, this nuclear protein was readily solubilised when nuclei were isolated and extracted sequentially with Triton, nucleases and salts. Despite this, nuclei containing pre-assembled replication forks could synthesise DNA when transferred into microcystin-treated extracts.
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Macejova, Dana, L. Toporova, and J. Brtko. "The role of retinoic acid receptors and their cognate ligands in reproduction in a context of triorganotin based endocrine disrupting chemicals." Endocrine Regulations 50, no. 3 (July 1, 2016): 154–64. http://dx.doi.org/10.1515/enr-2016-0018.
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Abstract Retinoic acid (RA), an active form of vitamin A, regulates the embryonic development, male and female reproduction and induces important effects on the cell development, proliferation, and differentiation. These effects are mediated by the retinoid (RAR) and rexinoid nuclear receptors (RXR), which are considered to be a ligand-activated, DNA-binding, trans-acting, and transcription-modulating proteins, involved in a general molecular mechanism responsible for the transcriptional responses in target genes. Organotin compounds are typical environmental contaminants and suspected endocrine disrupting substances. They may affect processes of reproductive system in mammals, predominantly via nuclear receptor signaling pathways. Triorganotins, such as tributyltin chloride (TBTCl) and triphenyltin chloride (TPTCl), are capable to bind to RXR molecules, and thus represent potent agonists of RXR subtypes of nuclear receptors not sharing any structural characteristics with endogenous ligands of nuclear receptors. Th is article summarizes selected effects of biologically active retinoids and rexinoids on both male and female reproduction and also deals with the effects of organotin compounds evoking endocrine disrupting actions in reproduction.
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Dabauvalle, M. C., K. Loos, H. Merkert, and U. Scheer. "Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin." Journal of Cell Biology 112, no. 6 (March 15, 1991): 1073–82. http://dx.doi.org/10.1083/jcb.112.6.1073.
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Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.
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Marcomini, Isabella, and Susan M. Gasser. "Nuclear organization in DNA end processing: Telomeres vs double-strand breaks." DNA Repair 32 (August 2015): 134–40. http://dx.doi.org/10.1016/j.dnarep.2015.04.024.
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Kubota, Y., and H. Takisawa. "Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts." Journal of Cell Biology 123, no. 6 (December 15, 1993): 1321–31. http://dx.doi.org/10.1083/jcb.123.6.1321.
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Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase-like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M-phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine-treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.
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Peng, Aimin, Andrea L. Lewellyn, and James L. Maller. "Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos." Molecular and Cellular Biology 27, no. 19 (July 30, 2007): 6852–62. http://dx.doi.org/10.1128/mcb.00195-07.
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ABSTRACT In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.
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Gronostajski, R. M., S. Adhya, K. Nagata, R. A. Guggenheimer, and J. Hurwitz. "Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites." Molecular and Cellular Biology 5, no. 5 (May 1985): 964–71. http://dx.doi.org/10.1128/mcb.5.5.964.
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Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.
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Gronostajski, R. M., S. Adhya, K. Nagata, R. A. Guggenheimer, and J. Hurwitz. "Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites." Molecular and Cellular Biology 5, no. 5 (May 1985): 964–71. http://dx.doi.org/10.1128/mcb.5.5.964-971.1985.
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Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.
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Jin, Pei, Stephen Hardy, and David O. Morgan. "Nuclear Localization of Cyclin B1 Controls Mitotic Entry After DNA Damage." Journal of Cell Biology 141, no. 4 (May 18, 1998): 875–85. http://dx.doi.org/10.1083/jcb.141.4.875.
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Mitosis in human cells is initiated by the protein kinase Cdc2-cyclin B1, which is activated at the end of G2 by dephosphorylation of two inhibitory residues, Thr14 and Tyr15. The G2 arrest that occurs after DNA damage is due in part to stabilization of phosphorylation at these sites. We explored the possibility that entry into mitosis is also regulated by the subcellular location of Cdc2-cyclin B1, which is suddenly imported into the nucleus at the end of G2. We measured the timing of mitosis in HeLa cells expressing a constitutively nuclear cyclin B1 mutant. Parallel studies were performed with cells expressing Cdc2AF, a Cdc2 mutant that cannot be phosphorylated at inhibitory sites. Whereas nuclear cyclin B1 and Cdc2AF each had little effect under normal growth conditions, together they induced a striking premature mitotic phenotype. Nuclear targeting of cyclin B1 was particularly effective in cells arrested in G2 by DNA damage, where it greatly reduced the damage-induced G2 arrest. Expression of nuclear cyclin B1 and Cdc2AF also resulted in significant defects in the exit from mitosis. Thus, nuclear targeting of cyclin B1 and dephosphorylation of Cdc2 both contribute to the control of mitotic entry and exit in human cells.
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Palacios-González, César. "Does egg donation for mitochondrial replacement techniques generate parental responsibilities?" Journal of Medical Ethics 44, no. 12 (October 25, 2017): 817–22. http://dx.doi.org/10.1136/medethics-2017-104400.
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Children created through mitochondrial replacement techniques (MRTs) are commonly presented as possessing 50% of their mother’s nuclear DNA, 50% of their father’s nuclear DNA and the mitochondrial DNA of an egg donor. This lab-engineered genetic composition has prompted two questions: Do children who are the product of an MRT procedure have three genetic parents? And, do MRT egg donors have parental responsibilities for the children created? In this paper, I address the second question and in doing so I also address the first one. First, I present a brief account of mitochondrial diseases and MRTs. Second, I examine how MRTs affect the numerical identity of eggs and zygotes. Third, I investigate two genetic accounts of parenthood and MRT egg donation. Fourth, I explore three causal accounts of parenthood and MRT egg donation. My conclusion is that, under the appropriate circumstances, MRT egg donors are parentally responsible for the children created under genetic accounts of parenthood and under causal accounts of parenthood.
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Pu, Robert T., and Mary Dasso. "The Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport." Molecular Biology of the Cell 8, no. 10 (October 1997): 1955–70. http://dx.doi.org/10.1091/mbc.8.10.1955.
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Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 fromXenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.
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Zhang, Guo-Jun, Ran Dong, Li-Na Lan, Shu-Fen Li, Wu-Jun Gao, and Hong-Xing Niu. "Nuclear Integrants of Organellar DNA Contribute to Genome Structure and Evolution in Plants." International Journal of Molecular Sciences 21, no. 3 (January 21, 2020): 707. http://dx.doi.org/10.3390/ijms21030707.
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The transfer of genetic material from the mitochondria and plastid to the nucleus gives rise to nuclear integrants of mitochondrial DNA (NUMTs) and nuclear integrants of plastid DNA (NUPTs). This frequently occurring DNA transfer is ongoing and has important evolutionary implications. In this review, based on previous studies and the analysis of NUMT/NUPT insertions of more than 200 sequenced plant genomes, we analyzed and summarized the general features of NUMTs/NUPTs and highlighted the genetic consequence of organellar DNA insertions. The statistics of organellar DNA integrants among various plant genomes revealed that organellar DNA-derived sequence content is positively correlated with the nuclear genome size. After integration, the nuclear organellar DNA could undergo different fates, including elimination, mutation, rearrangement, fragmentation, and proliferation. The integrated organellar DNAs play important roles in increasing genetic diversity, promoting gene and genome evolution, and are involved in sex chromosome evolution in dioecious plants. The integrating mechanisms, involving non-homologous end joining at double-strand breaks were also discussed.
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Hartl, P., E. Olson, T. Dang, and DJ Forbes. "Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate." Journal of Cell Biology 124, no. 3 (February 1, 1994): 235–48. http://dx.doi.org/10.1083/jcb.124.3.235.
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Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha-amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205-217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.
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Katsumata, M., and C. W. Lo. "Organization of chromosomes in the mouse nucleus: analysis by in situ hybridization." Journal of Cell Science 90, no. 2 (June 1, 1988): 193–99. http://dx.doi.org/10.1242/jcs.90.2.193.
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We used in situ hybridization with various biotinylated DNA probes to characterize the distribution of mouse chromosomes in the nucleus. This entailed an examination of plasmid-marked chromosomes from two strains of transgenic mice and also an examination of the distribution of centromeres and total mouse genomic DNA. Our results show that, regardless of the hybridization probes used or the tissue types examined, with sections of mouse tissues embedded in paraffin all chromosomal DNA appeared to be localized to the nuclear periphery. This was observed in tissues fixed with either crosslinking or non-crosslinking fixatives, and submitted to a variety of prehybridization treatments. Further experiments with non-embedded nuclei fractionated from mouse liver revealed a similar distribution of DNA at the telomeric end of chromosome 3, but for centromeric DNA and total mouse genomic DNA, hybridization signals were observed both in the interior and at the periphery of the nucleus. These observations preclude the exclusive localization of chromosomes to the nuclear periphery. However, they indicate that a subset of centromeres are likely to be associated with the nuclear membrane, and that there is such an association at the telomeric end of chromosome 3. Overall, these results are compatible with previous observations of other investigators showing regions of contact between chromosomes and the nuclear membrane.
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Safdar, Adeel, Jonathan P. Little, Andrew J. Stokl, Bart P. Hettinga, Mahmood Akhtar, and Mark A. Tarnopolsky. "Exercise Increases Mitochondrial PGC-1α Content and Promotes Nuclear-Mitochondrial Cross-talk to Coordinate Mitochondrial Biogenesis." Journal of Biological Chemistry 286, no. 12 (January 18, 2011): 10605–17. http://dx.doi.org/10.1074/jbc.m110.211466.
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Endurance exercise is known to induce metabolic adaptations in skeletal muscle via activation of the transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α). PGC-1α regulates mitochondrial biogenesis via regulating transcription of nuclear-encoded mitochondrial genes. Recently, PGC-1α has been shown to reside in mitochondria; however, the physiological consequences of mitochondrial PGC-1α remain unknown. We sought to delineate if an acute bout of endurance exercise can mediate an increase in mitochondrial PGC-1α content where it may co-activate mitochondrial transcription factor A to promote mtDNA transcription. C57Bl/6J mice (n = 12/group; ♀ = ♂) were randomly assigned to sedentary (SED), forced-endurance (END) exercise (15 m/min for 90 min), or forced endurance +3 h of recovery (END+3h) group. The END group was sacrificed immediately after exercise, whereas the SED and END+3h groups were euthanized 3 h after acute exercise. Acute exercise coordinately increased the mRNA expression of nuclear and mitochondrial DNA-encoded mitochondrial transcripts. Nuclear and mitochondrial abundance of PGC-1α in END and END+3h groups was significantly higher versus SED mice. In mitochondria, PGC-1α is in a complex with mitochondrial transcription factor A at mtDNA D-loop, and this interaction was positively modulated by exercise, similar to the increased binding of PGC-1α at the NRF-1 promoter. We conclude that in response to acute altered energy demands, PGC-1α re-localizes into nuclear and mitochondrial compartments where it functions as a transcriptional co-activator for both nuclear and mitochondrial DNA transcription factors. These results suggest that PGC-1α may dynamically facilitate nuclear-mitochondrial DNA cross-talk to promote net mitochondrial biogenesis.
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Manev, Hari, Svetlana Dzitoyeva, and Hu Chen. "Mitochondrial DNA: a blind spot in neuroepigenetics." BioMolecular Concepts 3, no. 2 (April 1, 2012): 107–15. http://dx.doi.org/10.1515/bmc-2011-0058.
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AbstractNeuroepigenetics, which includes nuclear DNA modifications, such as 5-methylcytosine and 5-hydroxymethylcytosine and modifications of nuclear proteins, such as histones, is emerging as the leading field in molecular neuroscience. Historically, a functional role for epigenetic mechanisms, including in neuroepigenetics, has been sought in the area of the regulation of nuclear transcription. However, one important compartment of mammalian cell DNA, different from nuclear DNA but equally important for physiological and pathological processes (including in the brain), mitochondrial DNA has for the most part not had a systematic epigenetic characterization. The importance of mitochondria and mitochondrial DNA (particularly its mutations) in central nervous system physiology and pathology has long been recognized. Only recently have the mechanisms of mitochondrial DNA methylation and hydroxymethylation, including the discovery of mitochondrial DNA-methyltransferases and the presence and functionality of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (e.g., in modifying the transcription of mitochondrial genome), been unequivocally recognized as a part of mammalian mitochondrial physiology. Here, we summarize for the first time evidence supporting the existence of these mechanisms and propose the term ‘mitochondrial epigenetics’ to be used when referring to them. Currently, neuroepigenetics does not include mitochondrial epigenetics – a gap that we expect to close in the near future.
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Fowler, Katie E., Anjali A. Mandawala, and Darren K. Griffin. "The role of chromosome segregation and nuclear organisation in human subfertility." Biochemical Society Transactions 47, no. 1 (February 7, 2019): 425–32. http://dx.doi.org/10.1042/bst20180231.
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Abstract Spermatogenesis is central to successful sexual reproduction, producing large numbers of haploid motile male gametes. Throughout this process, a series of equational and reductional chromosome segregation precedes radical repackaging of the haploid genome. Faithful chromosome segregation is thus crucial, as is an ordered spatio-temporal ‘dance’ of packing a large amount of chromatin into a very small space. Ergo, when the process goes wrong, this is associated with an improper chromosome number, nuclear position and/or chromatin damage in the sperm head. Generally, screening for overall DNA damage is relatively commonplace in clinics, but aneuploidy assessment is less so and nuclear organisation studies form the basis of academic research. Several studies have focussed on the role of chromosome segregation, nuclear organisation and analysis of sperm morphometry in human subfertility observing significant alterations in some cases, especially of the sex chromosomes. Importantly, sperm DNA damage has been associated with infertility and both extrinsic (e.g. lifestyle) and intrinsic (e.g. reactive oxygen species levels) factors, and while some DNA-strand breaks are repaired, unexpected breaks can cause differential chromatin packaging and further breakage. A ‘healthy’ sperm nucleus (with the right number of chromosomes, nuclear organisation and minimal DNA damage) is thus an essential part of reproduction. The purpose of this review is to summarise state of the art in the fields of sperm aneuploidy assessment, nuclear organisation and DNA damage studies.
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Leno, G. H., and R. A. Laskey. "The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts." Journal of Cell Biology 112, no. 4 (February 15, 1991): 557–66. http://dx.doi.org/10.1083/jcb.112.4.557.
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We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.
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Zheng, Li, Yuan Meng, Judith L. Campbell, and Binghui Shen. "Multiple roles of DNA2 nuclease/helicase in DNA metabolism, genome stability and human diseases." Nucleic Acids Research 48, no. 1 (November 22, 2019): 16–35. http://dx.doi.org/10.1093/nar/gkz1101.
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Abstract DNA2 nuclease/helicase is a structure-specific nuclease, 5′-to-3′ helicase, and DNA-dependent ATPase. It is involved in multiple DNA metabolic pathways, including Okazaki fragment maturation, replication of ‘difficult-to-replicate’ DNA regions, end resection, stalled replication fork processing, and mitochondrial genome maintenance. The participation of DNA2 in these different pathways is regulated by its interactions with distinct groups of DNA replication and repair proteins and by post-translational modifications. These regulatory mechanisms induce its recruitment to specific DNA replication or repair complexes, such as DNA replication and end resection machinery, and stimulate its efficient cleavage of various structures, for example, to remove RNA primers or to produce 3′ overhangs at telomeres or double-strand breaks. Through these versatile activities at replication forks and DNA damage sites, DNA2 functions as both a tumor suppressor and promoter. In normal cells, it suppresses tumorigenesis by maintaining the genomic integrity. Thus, DNA2 mutations or functional deficiency may lead to cancer initiation. However, DNA2 may also function as a tumor promoter, supporting cancer cell survival by counteracting replication stress. Therefore, it may serve as an ideal target to sensitize advanced DNA2-overexpressing cancers to current chemo- and radiotherapy regimens.
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Weaver, V. M., C. E. Carson, P. R. Walker, N. Chaly, B. Lach, Y. Raymond, D. L. Brown, and M. Sikorska. "Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes." Journal of Cell Science 109, no. 1 (January 1, 1996): 45–56. http://dx.doi.org/10.1242/jcs.109.1.45.
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In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occurred within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferase-mediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.
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O'Brien, Lori L., and Christiane Wiese. "TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts." Journal of Cell Biology 173, no. 5 (May 30, 2006): 685–94. http://dx.doi.org/10.1083/jcb.200512107.
Full textAbstract:
Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.
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May, Bernie, and Peter M. Grewe. "Fate of maternal mtDNA following 60Co inactivation of maternal nuclear DNA in unfertilized salmonid eggs." Genome 36, no. 4 (August 1, 1993): 725–30. http://dx.doi.org/10.1139/g93-097.
Full textAbstract:
The effects of gamma irradiation on nuclear DNA and mitochondrial DNA (mtDNA) were examined by exposing unfertilized salmonid eggs to a 60Co source. Brown trout (Salmo trutta) eggs exposed to 60Co were fertilized with sperm from brook trout (Salvelinus fontinalis), and brook trout eggs exposed to 60Co were fertilized with sperm from splake males (S. namaycush × S. fontinalis). In both types of matings only paternal allozymes were found in embryos, confirming the inactivation of the nuclear genome in the eggs. Analysis of mtDNA in these same embryos showed exclusively maternal mtDNA. The absence of paternal mtDNA among any of the embryos supports the predominance of maternal inheritance of mtDNA in vertebrates and suggests that mtDNAs are more resistant to cobalt inactivation than nuclear DNAs based on structure or numerical superiority to maternal nuclear DNA. Inactivation of maternal nuclear DNA, fertilization, and an induced return to the diploid state provide a means for producing an inbred organism having the nuclear genome of the paternal parent (androgenetic) and the mitochondrial genome of the female.Key words: mtDNA, maternal transmission, salmonid, egg, 60Co.
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Lawlis, S. J., S. M. Keezer, J. R. Wu, and D. M. Gilbert. "Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts." Journal of Cell Biology 135, no. 5 (December 1, 1996): 1207–18. http://dx.doi.org/10.1083/jcb.135.5.1207.
Full textAbstract:
Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.
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Kase, Julia, Hua Jing, Jan R. Dörr, Maja Milanovic, Dido Lenze, Michael Grau, Gregor Beuster, et al. "Contrary Functions of NF-κB Signaling in Anti-Cancer Treatment Outcome." Blood 118, no. 21 (November 18, 2011): 1652. http://dx.doi.org/10.1182/blood.v118.21.1652.1652.
Full textAbstract:
Abstract Abstract 1652 Nuclear factor-κB (NF-κB) transcription factors are involved in cancer-relevant processes such as suppression of apoptosis, growth promotion, enhanced migration and invasiveness, although their actual role as oncogenic or tumor-suppressive activities remains controversial. Moreover, NF-κB-mediated suppression of apoptosis has been linked to chemoresistance. Interestingly, cellular senescence, a terminal cell-cycle arrest initiated via DNA-damaging chemotherapy as well, and known to improve long-term outcome, is associated with the massive induction of secretable NF-κB target genes, which, in turn, potentially reinforce the senescence phenotype. In this study, primary Eμ-myc transgenic mouse lymphomas as a well established model for human aggressive B-NHL, and information from human diffuse large B-cell lymphomas (DLBCL) were used in a cross-species approach to identify oncogenic networks in which chemotherapy-activated NF-κB signaling no longer mediates resistance but promotes therapy-induced senescence (TIS) and contributes to the outcome of anti-cancer treatment. Primary Myc-driven lymphoma cells, stably bcl2-transduced to block apoptosis, were exposed to the DNA-damaging chemotherapeutic agent adriamycin (ADR) to induce TIS. Gene set enrichment analysis of microarray-based gene expression profiles from drug-senescent vs. untreated cells found NF-κB target genes strongly skewed towards the TIS group, and multiplex ELISA-based analysis detected significantly higher DNA binding activities for the NF-κB family subunits p50, p52, p65 (RelA) and RelB in senescent cells. Inactivation of NF-κB by stable expression of the NF-κB super-repressor IκBα-δN (SR) lowered expression levels of NF-κB target genes in ADR-treated lymphomas. Matched pairs of individual primary lymphomas differing only in their SR status displayed compromised senescence induction in vivo when expressing the SR, indicating that TIS depends on intact NF-κB function. To assess the contribution of endogenous NF-κB signaling to long-term outcome, we grouped primary Myc-lymphomas by their NF-κB activity levels as “NF-κB low” (NL) or “NF-κB high” (NH). ADR-treated NH, but not NL lymphomas presented selective vulnerability to the SR moiety. Recapitulating the clinical outcome of patients suffering from DLBCL, around 60% of the mice harboring Eμ-myc lymphomas achieved long-term remissions, while the remaining 40% encountered a relapse after chemotherapy. Relapsing Myc-lymphomas exhibited substantially higher expression of the NF-κB targets IκBα and bcl2, reminiscent of activated B-cell-like (ABC) DLBCL, the clinically inferior subtype characterized by constitutively active NF-κB signaling. In contrast, germinal center B-cell-like (GCB) DLBCL rarely possess activating NF-κB mutations, but frequently develop in the context of a t(14;18) translocation that drives Bcl2 overexpression independent of NF-κB. Induced NF-κB target gene expression and increased TIS induction after overexpression of an NF-κB-activating CARD11 mutant suggested that higher NF-κB activity may contribute to treatment outcome via TIS promotion. Indeed, stratifying a large dataset of untreated GEP and corresponding clinical data after immunochemotherapy from 233 DLBCL patients by a 63-gene NF-κB expression signature (Shaffer-AL et al, Immunol Rev, 2006; Lenz-G et al, N Eng J Med, 2008), confirmed for the subset of GCB patients with above-median Bcl2 expression – the group whose genetic features we modeled before in the mouse – a significantly superior progression-free survival. In essence, our “cross-species” investigations demonstrate opposing roles of NF-κB in treatment outcome and have important ramifications for clinical trials that aim at inhibiting NF-κB signaling in DLBCL. Disclosures: No relevant conflicts of interest to declare.
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Tremblay, Maxime, Martin Toussaint, Annie D’Amours, and Antonio Conconi. "Nucleotide excision repair and photolyase repair of UV photoproducts in nucleosomes: assessing the existence of nucleosome and non-nucleosome rDNA chromatin in vivoThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process." Biochemistry and Cell Biology 87, no. 1 (February 2009): 337–46. http://dx.doi.org/10.1139/o08-128.
Full textAbstract:
The genome is organized into nuclear domains, which create microenvironments that favor distinct chromatin structures and functions (e.g., highly repetitive sequences, centromeres, telomeres, noncoding sequences, inactive genes, RNA polymerase II and III transcribed genes, and the nucleolus). Correlations have been drawn between gene silencing and proximity to a heterochromatic compartment. At the other end of the scale are ribosomal genes, which are transcribed at a very high rate by RNA polymerase I (~60% of total transcription), have a loose chromatin structure, and are clustered in the nucleolus. The rDNA sequences have 2 distinct structures: active rRNA genes, which have no nucleosomes; and inactive rRNA genes, which have nucleosomes. Like DNA transcription and replication, DNA repair is modulated by the structure of chromatin, and the kinetics of DNA repair vary among the nuclear domains. Although research on DNA repair in all chromosomal contexts is important to understand the mechanisms of genome maintenance, this review focuses on nucleotide excision repair and photolyase repair of UV photoproducts in the first-order packing of DNA in chromatin: the nucleosome. In addition, it summarizes the studies that have demonstrated the existence of the 2 rDNA chromatins, and the way this feature of the rDNA locus allows for direct comparison of DNA repair in 2 very different structures: nucleosome and non-nucleosome DNA.