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Untersuchungen über den Origin Recognition Complex (ORC) und über den Replikationsinitiator Cdc6p

Untersuchungen über den Origin Recognition Complex (ORC) und über den Replikationsinitiator Cdc6p

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BIERMANN, Esther, 2004. Untersuchungen über den Origin Recognition Complex (ORC) und über den Replikationsinitiator Cdc6p

@phdthesis{Biermann2004Unter-8035, title={Untersuchungen über den Origin Recognition Complex (ORC) und über den Replikationsinitiator Cdc6p}, year={2004}, author={Biermann, Esther}, address={Konstanz}, school={Universität Konstanz} }

Biermann, Esther Biermann, Esther 2004 Untersuchungen über den Origin Recognition Complex (ORC) und über den Replikationsinitiator Cdc6p In the first part of the available thesis the DNA-binding of the human ORC complex was characterized more exactly by in vitro studies. The following ORC complexes could be expressed and up-cleaned by the Baculovirus system: ORC2-5, ORC1-5 and ORC1-5/Cdc6. The binding activity of these complexes was analyzed in gel-shift-experiments and target-bound-assays with the UPR-fragment from the MCM4-promotor region and a pUPR-plasmid, which contains also the UPR-region. It was stated that, differently than in low eukaryotes such as Saccharomyces cerevisiae, the human ORC proteins bind nonspecific to DNA, whereby a preference for AT-rich sequences could be identified. In addition could be shown that the ORC1-5- and ORC1-5/Cdc6-complex obtained one, probably by Orc1 and Cdc6, stronger DNA connection exhibits than the ORC2-5-complex. Divide this result agree with observations in Xenopus laevis.<br />In the following parts of the work through in vivo studies the human replication protein Cdc6 was characterized during the cell cycle and the function was examined by the cell-free in vitro replication-system.<br />It was found that Cdc6 is synthesized to the same quantities during the cell cycle. This information increases the growing realizations over the expression of Cdc6 in mammalian cells. The sequential and continuous synthesis of Cdc6 is surprising in S-phase. The subcellular distribution of Cdc6 during the cell cycle is steered mainly by phosphorylation, phosphorylated Cdc6 becomes detached in S phase and is transported to the cytoplasm. Despite S phase mediated core-cytoplasm-transfer some Cdc6 still remains chromatin-bound. A reason for this could be the high quantity forming of Cdc6 during S phase.<br />In addition in this section it was shown that the quantities of marked chromatin-bound Cdc6 are similar in G1 and S phase. These results prove that Cdc6 dissociates during S phase of the chromatin, is transferred to the cytoplasm and is then replaced by newly synthesized Cdc6. Once bound to chromatin, the again marked Cdc6 behaves like the old one; it dissociates and disappears from the chromatin with a radioactive half-life of < 4h. This cycle of synthesis, chromatin binding and dissociation must be connected with functions of Cdc6 in the genome-replication. One function is surely the assembly of the pre-replication-complex, but still further functions must be necessary for the initiation of the DNA-replication or the time after, e.g. the activation of late origins.<br /><br />The data of this work inform additionally to already existing results about components and structure of the pre-replication-complexes and can therefore be used as basis for developing studies. 2011-03-24T17:39:25Z Investigations about the origin recognition complex (ORC) and the replication initiator Cdc6p deu deposit-license application/pdf 2011-03-24T17:39:25Z

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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