Article

In vitro replication of plasmids containing human c-myc DNA.

Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH 45435.
Journal of Molecular Biology (Impact Factor: 3.96). 02/1995; 245(2):92-109. DOI: 10.1006/jmbi.1994.0010
Source: PubMed

ABSTRACT A chromosomal replication initiation zone was previously mapped in cell cultures to the 5' flanking DNA of the human c-myc gene. We have used an in vitro system to examine the replication of a plasmid, pNeo.Myc-2.4, containing 2.4 kb of the c-myc initiation zone. In vitro, pNeo.Myc-2.4 generated high levels of DpnI-resistant DNA above background incorporation into control plasmids. pNeo.Myc-2.4 replicated semiconservatively to produce supercoiled and relaxed plasmid monomers, and replicative intermediates. [32P]dCMP incorporated into pNeo.Myc-2.4 appeared in Okazaki fragments and low molecular weight strands which matured to full length plasmid DNA, whereas [32P]dCMP incorporated into control plasmids appeared as continuous smears on denaturing gels. Other assays also distinguished the processive replication of pNeo.Myc-2.4 from the dispersive labeling of control plasmids. A pNeo.Myc-2.4 replication time course showed a clear preference for initiation within a restriction fragment containing the c-myc DNA. Two-dimensional electrophoresis revealed that a restriction fragment bearing the c-myc origin zone generated an arc characteristic of replicative intermediates containing a central replication bubble, while vector fragments in the plasmid generated arcs of forked intermediates. Replication bubbles visualized by electron microscopy were centered within the replication initiation zone, approximately 1.4 kb upstream of c-myc promoter P1. Okazaki fragments radiolabeled during in vitro replication showed a switch in the asymmetry of template preference within the initiation zone identified by electron microscopy, two-dimensional electrophoresis and early labeling. These data show that bidirectional, semiconservative replication can originate preferentially in vitro in the 5' flanking DNA of the c-myc gene, and that replicative intermediates present at low levels can be distinguished from molecules generated by competing, repair-type processes.

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Die Ergebnisse, die im zweiten Teil beschrieben wurden, haben gezeigt, dass das Plasmid pEPI-1 in CHO- und in humanen HeLa-Zellen extrachromosomal vorliegt, genau einmal pro Zellzyklus repliziert wird und dass die Replikation sehr früh in der S-Phase des wirtseigenen Zellzyklus erfolgt. Das episomale Replikon bleibt über mehrere Generationen ohne Selektionsdruck stabil in den Zellen vorhanden, was auf eine erfolgreiche Segregation während der Mitose zurückzuführen ist. Zudem konnte gezeigt werden, dass Komponenten des Prä-Replikationskomplexes an pEPI-1 in vivo binden. Die Beobachtung, dass das Bindungsverhalten und die Zusammen-setzung der untersuchten Replikations-Initiationsfaktoren zellzyklus-abhängig sind, deutet darauf hin, dass die Prä-Replikationskomplexe an pEPI-1 funktionsfähig sind. Mcm3p und Orc1p haben sich im Zuge der S-Phase von pEPI-1 abgelöst und damit eine vergleichbare Dynamik im Bindungsverhalten gezeigt wie sie bei zellulären Prä-Replikationskomplexen beobachtet wurde. Zudem hat ein durchgeführter „nascent DNA strand abundance“-Assay ergeben, dass die Initiation der DNA-Replikation an mehreren Stellen des pEPI-1-Plasmides erfolgen kann. Derivate von pEPI-1, bei denen die Sequenz einer bekannten humanen ORC-Bindestelle einkloniert wurde, haben weiterhin eine gleichmäßige, über das Plasmid verteilte ORC-Bindung gezeigt. Die Schlussfolgerung aus diesen Studien ist, dass die DNA-Sequenz für die Definition einer ORC-Bindestelle bei höheren Eukaryoten eine untergeordnete Rolle spielt und dass bei Säugerzellen viel mehr epigenetische Faktoren den Ort der ORC-Bindung und damit die Startstelle der DNA-Replikation bestimmen. Im dritten Teil dieser Arbeit wurde die Entwicklung und Charakterisierung eines zellfreien in vitro Replikationssystemes aus humanen Zellextrakten dargestellt. Es konnte gezeigt werden, dass eine Depletion von menschlichen Orc- oder Mcm-Proteinen zu einer vollständigen Inhibition der in vitro Replikation führt. Dies deutet darauf hin, dass Replikations-Initiationsfaktoren ein essentieller Bestandteil von diesem System sind. Im letzten Teil dieser Arbeit ist es schließlich gelungen, menschliche Mcm-Proteine mit Hilfe des eukaryotischen Baculovirus-Expressionssystemes herzustellen und unterschiedliche MCM-Proteinkomplexe zu isolieren. Ein trimerer hMcm4/6/7p-Komplex hat jedoch entgegen einer Reihe von Veröffentlichungen weder ATPase- noch Helikase-Aktivität gezeigt. Auch konnte keine Affinität zu einzelsträngiger DNA beobachtet werden. Im Zuge der biochemischen Charakterisierung eines gereinigten hexameren hMcm2-7p-Komplexes wurde dessen Bindungsaffinität zu doppel-strängige DNA festgestellt. In this work I have investigated the molecular mechanisms of the initiation of DNA-replication by in vivo and in vitro studies. 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. 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. 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. 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.