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Items 56 to 60 of 3044 total

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  • Defining Multiple Steps in Human Telomere End Processing

    Chow, THT;
    Telomere overhangs are essential for chromosome end protection and telomerase extension, but how telomere overhangs are generated is unknown. Due to the classic end replication problem, leading DNA daughter strands are initially blunt while lagging daughters are shorter by at least the size of the final RNA primer, which historically is believed to be located at extreme chromosome ends. We developed a variety of new approaches to define the steps in the processing of these overhangs. Understanding the number and nature of the overhang processing events is crucial in establishing the roles of candidate proteins involved. We here define these steps in normal human cells. We show the final lagging RNA primer is positioned ~70-100 nt from chromosome ends (not at the extreme ends), and is not removed for ~1hr following replication. Therefore, the location of the RNA primer, rather than its size, is a primary driving force for telomere shortening. Moreover, we demonstrate that telomere end-processing occurs in two distinct phases following telomere duplex replication. During the early phase, which occupies 1-2 hours following telomere replication, several steps occur on both leading and lagging daughters. Leading telomere processing remains incomplete until late S/G2 when the C-terminal nucleotide is specified, referred to as the late phase. Furthermore, in human cancer cells under maintenance condition, telomerase extension is uncoupled from C-strand fill-in. These results uncover crucial mechanistic details of the DNA end-replication problem as well as several specific steps in telomere overhang processing. These results also indicate the presence of previously unsuspected complexes and signaling events required for the replication of the ends of human chromosomes. The findings and the methods developed will now provide the basis for examining candidate factors that may function to regulate particular steps in telomere length homeostasis with implications in both cellular aging and cancer.
  • Zellteilungsregulation meristematischer Wurzelzellen (Tabak BY-2) durch Phytohormone und Zucker

    Hartig, K;
    Die Phytohormone Cytokinin und Auxin sind essentiell für die Zellteilungsaktivität in meristematischen Geweben. Trotz ihrer zentralen Bedeutung im pflanzlichen Zellzyklus, ist über deren Bedeutung und Wirksamkeit auf molekularer Ebene bisher nur wenig bekannt. Einige Untersuchungen deuten darauf hin, dass mit Hilfe hormoneller Signale die Zellteilungsaktivität der Meristeme an die physiologischen Gegebenheiten der gesamten Pflanze angepasst wird, und auf diese Weise der Verbrauch an Zucker, die sogenannte Sinkstärke des Meristems definiert wird. Besonders bedeutsam ist deshalb der "cross-talk" der hormonellen Signale mit den metabolischen Signalen. Da diese Signalverknüpfung bisher kaum Ziel wissenschaftlicher Untersuchungen war, sollte diese Arbeit zur Aufklärung betragen. Die Untersuchungen wurden mit einer Tabak Zellkultur (BY-2) durchgeführt, die als Modell eines Wurzelmeristems dienen sollte. Ein Vorteil dieser Zellkultur ist die Möglichkeit, den Zellzyklus der Zellen durch auswaschbare Hemmstoffe synchronisieren zu können. Diese Synchronisierung eröffnet die Möglichkeit, die Effekte von Cytokininen, Auxinen und Zucker auf den Ablauf des Zellzyklus mittels Durchflusscytometrie untersuchen zu können, in dem Qualität und Quantität dieser Faktoren variiert wurde. Parallel dazu wurde die Expression von Zellzyklus-Kontrollgenen mittels semiquantitativer RT-PCR analysiert und entsprechend responsive Gene identifiziert. Ergänzt wurden die Untersuchen der Regulation des Zellzyklus durch längerfristige Beobachtungen der Entwicklung der Zellzahl, des Frischgewichtes und des Volumens der Zellen. Zur Charakterisierung der Signalentstehung, wurden die extrazellulären und intrazellulären Konzentrationen der die Zellteilung steuernden Faktoren gemessen.
  • Genetic analysis of the Replication Protein A large subunit family in Arabidopsis reveals unique and overlapping roles in DNA repair, meiosis and DNA replication

    Aklilu, BB; Soderquist, RS; Culligan, KM;
    Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH 03824, USA and Program in Genetics, University of New Hampshire, Durham NH 03824, USA
    Replication Protein A (RPA) is a heterotrimeric protein complex that binds single-stranded DNA. In plants, multiple genes encode the three RPA subunits (RPA1, RPA2 and RPA3), including five RPA1-like genes in Arabidopsis. Phylogenetic analysis suggests two distinct groups composed of RPA1A, RPA1C, RPA1E (ACE group) and RPA1B, RPA1D (BD group). ACE-group members are transcriptionally induced by ionizing radiation, while BD-group members show higher basal transcription and are not induced by ionizing radiation. Analysis of rpa1 T-DNA insertion mutants demonstrates that although each mutant line is likely null, all mutant lines are viable and display normal vegetative growth. The rpa1c and rpa1e single mutants however display hypersensitivity to ionizing radiation, and combination of rpa1c and rpa1e results in additive hypersensitivity to a variety of DNA damaging agents. Combination of the partially sterile rpa1a with rpa1c results in complete sterility, incomplete synapsis and meiotic chromosome fragmentation, suggesting an early role for RPA1C in promoting homologous recombination. Combination of either rpa1c and/or rpa1e with atr revealed additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways. In contrast, rpa1b rpa1d double mutant plants display slow growth and developmental defects under non-damaging conditions. We show these defects in the rpa1b rpa1d mutant are likely the result of defective DNA replication leading to reduction in cell division.
    10.1093/nar/gkt1292
  • Genetic and evolutionary analysis of plant replication protein A 1 (RPA1)

    Aklilu, BB;
    Challenging human health issues include treatments for genetic diseases and providing improved agricultural crop output to feed the growing world population. The project described here, which focuses on how cells respond to chromosomal (genomic) damage, has significant implications in each example. In humans, accumulation of DNA damage induced mutations can result in genetic diseases such as cancer, and in plants can similarly result in genome instability, reducing productivity. Organisms from human to plants have conserved mechanisms to counteract DNA damage. However, detailed genetic and biochemical information on plant DNA repair systems is still limited. The goal of this dissertation was to characterize the role of the Replication Protein A1 (RPA1) genes in DNA repair and related cellular processes such as DNA replication and meiotic recombination in Arabidopsis thaliana.

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