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An episomal mammalian replicon: sequence‐independent binding of the origin recognition complex
Author(s) -
Schaarschmidt Daniel,
Baltin Jens,
Stehle Isa M,
Lipps Hans J,
Knippers Rolf
Publication year - 2004
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1038/sj.emboj.7600029
Subject(s) - biology , origin recognition complex , replicon , plasmid , chromatin , origin of replication , dna replication , dna , microbiology and biotechnology , extrachromosomal dna , eukaryotic dna replication , control of chromosome duplication , minichromosome maintenance , genetics
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.