Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells.
Origins of replication are expected to recruit initiation proteins like origin recognition complex (ORC) and Cdc6 in eukaryotes and provide a platform for unwinding DNA. Here we test whether localization of initiation proteins onto DNA is sufficient for origin function. Different components of the ORC complex and Cdc6 stimulated prereplicative complex (pre-RC) formation and replication initiation when fused to the GAL4 DNA-binding domain and recruited to plasmid DNA containing a tandem array of GAL4-binding sites. Replication occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle. The GAL4 fusion protein recruits other polypeptides of the ORC-Cdc6 complex, and nascent strand abundance was highest near the GAL4-binding sites. Therefore, the artificial origin recapitulates many of the regulatory features of physiological origins and is valuable for studies on replication initiation in mammalian cells. We demonstrated the utility of this system by showing the functional importance of the ATPase domains of human Cdc6 and Orc1 and the dispensability of the N-terminal segments of Orc1 and Orc2 in this assay. Artificial recruitment of a eukaryotic cellular replication initiation factor to a DNA sequence can create a functional origin of replication, providing a robust genetic assay for these factors and a novel approach to generating episomal vectors for gene therapy.
Takeda
David Y
DY
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Shibata
Yoshiyuki
Y
Parvin
Jeffrey D
JD
Dutta
Anindya
A
D000251:Adenosine Triphosphatases; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D051738:Origin Recognition Complex / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
C502132:CDC6 protein, mouse; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D000251:Adenosine Triphosphatases