Biochemische Studien über eukaryotische Replikations-Initiationsfaktoren

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SCHAARSCHMIDT, Daniel, 2004. Biochemische Studien über eukaryotische Replikations-Initiationsfaktoren

@phdthesis{Schaarschmidt2004Bioch-6774, title={Biochemische Studien über eukaryotische Replikations-Initiationsfaktoren}, year={2004}, author={Schaarschmidt, Daniel}, address={Konstanz}, school={Universität Konstanz} }

deposit-license 2011-03-24T17:29:05Z deu In this work I have investigated the molecular mechanisms of the initiation of DNA-replication by in vivo and in vitro studies.<br />In the first part of this work I could show that human MCM proteins appear to assemble in vivo around bound ORC proteins. I investigated the loading of the MCM proteins by a procedure that involved in vivo formaldehyde crosslinking and chomatin immunoprecipitation (ChIP). MCM binding sites were detected close to established ORC binding sites and start sites of replication in the upstream promoter regions of two human genes. I showed that MCM-specific antibodies preferentially precipitate these origin-containing sequences from G1 phase cells, but not from S phase cells where MCM proteins appear to be distributed over the entire chromatin sections investigated.<br />The results from the second part of this work showed that the plasmid pEPI-1 replicates as an episomal element in cultured CHO or HeLa cells and is replicated in an once-per-cell-cycle manner early in S phase. 5-10 copies per cell are stably maintained for more than 100 generations in the absence of selection pressure and in the absence of virally encoded transactivating factors. Chromatin immunoprecipitations and quantitative PCR analysis revealed that, in G1 phase cells, Orc1 and Orc2, as well as Mcm3, another component of the pre-replication complex, are bound to multiple sites on the plasmid. These binding sites are functional because they show the S-phase-dependent dissociation of Orc1 and Mcm3 known to be characteristic for pre-replication complexes in mammalian cells. In addition, we identified replicative nascent strands and showed that they correspond to many plasmid DNA regions. This work has implications for current models of replication origins in mammalian systems. It indicates that specific DNA sequences are not required for the chromatin-binding of ORC in vivo. The conclusion is that epigenetic mechanisms determine the sites where mammalian DNA replication is initiated.<br />In the third part of this work I developed and characterized a cell-free in vitro DNA-replication assay from human cells. I could show that the depletion of human ORC or MCM proteins leads to an inhibition of the in vitro replication of DNA.<br />The expression of recombinant human MCM-proteins was the aim of the last part of this work. Using the baculovirus expression system I could isolate a MCM4/6/7 complex which was lacking any ATPase- or helicase-activity. This complex showed also no DNA-binding activity. Studying the biochemistry of a hexameric MCM2-7 complex I detected binding activity to double stranded DNA. Schaarschmidt, Daniel Schaarschmidt, Daniel 2011-03-24T17:29:05Z 2004 Biochemical studies on eucaryotic replication initiation factors Biochemische Studien über eukaryotische Replikations-Initiationsfaktoren application/pdf

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