An episomal mammalian replicon: sequence-independent binding of the origin recognition complex.
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.
Schaarschmidt
Daniel
D
Department of Biology, Universität Konstanz, Konstanz, Germany. daniel.schaarschmidt@uni-konstanz.de
Baltin
Jens
J
Stehle
Isa M
IM
Lipps
Hans J
HJ
Knippers
Rolf
R
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008322:Mammals / Q000235:genetics*; D045170:Matrix Attachment Regions; D064126:Minichromosome Maintenance Complex Component 3; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012093:Replicon
D018797:Cell Cycle Proteins; D002843:Chromatin; D004270:DNA, Circular; D004268:DNA-Binding Proteins; C066470:MCM3 protein, human; D009687:Nuclear Proteins; C497223:ORC1 protein, human; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D064126:Minichromosome Maintenance Complex Component 3