An episomal mammalian replicon: sequence‐independent binding of the origin recognition complex

• Published in: The EMBO journal Vol. 23; no. 1; pp. 191 - 201
• Main Authors: Schaarschmidt, Daniel, Baltin, Jens, Stehle, Isa M, Lipps, Hans J, Knippers, Rolf
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 14.01.2004; Blackwell Publishing Ltd
• Subjects: Animals; Base Pairing; Base Sequence; Cell Cycle Proteins - metabolism; CHO Cells; Chromatin - metabolism; Clone Cells; Cricetinae; Cricetulus; Deoxyribonucleic acid; DNA; DNA, Circular - chemistry; DNA, Circular - metabolism; DNA-Binding Proteins - metabolism; HeLa Cells; Humans; initiation of DNA replication; mammalian episome; Mammals; Mammals - genetics; Matrix Attachment Regions; Mcm3 protein; Minichromosome Maintenance Complex Component 3; Nuclear Proteins; Orc1 protein; Orc2 protein; origin of replication; Origin Recognition Complex; Plasmids; Polymerase Chain Reaction; Precipitin Tests; Promoter Regions, Genetic; Replicon
• DOI: 10.1038/sj.emboj.7600029
• ISSN: 0261-4189

An extrachromosomally replicating plasmid was used to investigate the specificity by which the origin recognition complex (ORC) interacts with DNA sequences in mammalian cells in vivo. We first showed that the plasmid pEPI‐1 replicates semiconservatively in a once‐per‐cell‐cycle manner and is stably transmitted over many cell generations in culture without selection. Chromatin immunoprecipitations and quantitative polymerase chain reaction analysis revealed that, in G1‐phase cells, Orc1 and Orc2, as well as Mcm3, another component of the prereplication 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 prereplication 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.