Chromatin-associated Proteins in Cultured Human Cells

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KREITZ, Sandra, 2002. Chromatin-associated Proteins in Cultured Human Cells [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Kreitz2002Chrom-8514, title={Chromatin-associated Proteins in Cultured Human Cells}, year={2002}, author={Kreitz, Sandra}, address={Konstanz}, school={Universität Konstanz} }

application/pdf eng Chromatin-associated Proteins in Cultured Human Cells 2011-03-24T17:44:18Z Kreitz, Sandra deposit-license Kreitz, Sandra Chromatin-assoziierte Proteine in kultivierten menschlichen Zellen For the expression and replication of the eukaryotic genome, structural as well as regulatory proteins are indispensable. This thesis deals with the characterization of an example of both categories: the Ki-67 protein involved in the organization of heterochromatin and Orc1, a protein essential for the initiation of DNA replication. (Part I) The Ki-67 protein: an example for a structural nuclear protein. The nuclear protein Ki-67 is tightly associated with cell proliferation and therefore used as a prognostic proliferation marker in histopathology. Its functional significance, however, has still not been clarified completely. Application of cell fractionation and nuclease digestion experiments in this study revealed that Ki-67p resides at nuclear sites that are sensitive to moderately high salt concentrations and hardly accessible to nucleases under isotonic conditions. However, chromatin prepared under very low ionic strength was more accessible and digestion with micrococcal nuclease released DNA fragments carrying Ki-67p. Results presented in this thesis demonstrate for the first time that Ki-67p is bound to DNA in vivo. (to be continued below)<br />(Part I, see above) (Part II) The Orc1 protein: an example for a replication initiation protein. The precisely coordinated replication of the eukaryotic genome requires several key proteins involved in the initiation of replication. This thesis deals with the characterization of one of these factors, the human ORC complex. Consisting of six proteins, hOrc1p-hOrc6p, ORC recognizes replication origins on chromatin and recruits additional factors to eventually assemble the replication machinery at these sites. Investigation of the composition of ORC in HeLa cells revealed the existence of two major forms of ORC, one comprised of hOrc1p-hOrc5p and one that lacks hOrc1p. Both forms differ in their binding properties to nuclear sites. Functional differences of both ORC forms become evident upon passage through S-phase when the hOrc1p-containing complexes are destabilized probably due to the dissociation of hOrc1p from chromatin. Evidence is provided that the release of hOrc1p is accompanied by its phosphorylation and depends on progression of replication forks. Further experiments suggest that the released hOrc1p is degraded by the 26S-proteasome. Thus, the cell cycle-regulated association of hOrc1p with DNA might represent a new mechanism to limit initiation at each origin to once per cell cycle. 2002 2011-03-24T17:44:18Z

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