Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

Abstract

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. In order to duplicate the massive eukaryotic genome in a reasonable amount of time, DNA replication begins from multiple sites located throughout the genome called replication origins (for review, see Gilbert 2001). In the budding yeast, Saccharomyces cerevisiae, replication origins are defined by specific DNA sequences ∼100 base pairs (bp) in length. Plasmids containing these sequences replicate when transformed into cells and hence are referred to as autonomously replicating sequences, or ARS. Within each ARS is an 11-bp ARS consensus sequence (ACS) that is required for binding of the six-subunit origin recognition complex (ORC). ORC remains bound to chromatin throughout the cell cycle in S. cerevisiae, where it catalyzes the ordered assembly of replication factors that culminate in the loading of the replicative polymerases that begin DNA synthesis (Bell and Dutta 2002;Mendez and Stillman 2003). Although ORC and many of the proteins involved in replication are conserved in all eukaryotes, identification of conserved DNA sequences that define eukaryotic origins remains elusive (Gilbert 2004;Cvetic and Walter 2005). Similar plasmid transformation experiments that were successful in budding yeast have failed to identify ARS sequences in other eukaryotes. In mammalian cells, replication initiation occurs from poorly localized replication zones of 6-55 kb, although in a few cases the origin has been mapped to smaller lengths of DNA. Having welldefined origins in S. cerevisiae has greatly facilitated studying replication initiation. The initial discovery and characterization of ORC w...

Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins

“…4D). This study is not the first example that origins can be specified on plasmid DNAs (32,33). However, a direct interaction between ORC and a chromatin constituent like HMGA1a in vivo, and a possible contribution to origin formation has not been described before.…”

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

“…In yeast its conditional knockout causes the lack of pre-RC formation (8), and the knockdown by RNAi of human CDC6 prevents cell proliferation and induces apoptosis (18,37). Moreover, the targeting of CDC6 to a plasmid has been shown to be sufficient to generate an artificial replication origin (58). The expression of HOXD13 within cells has a critical influence on the timing of origin licensing and of the initiation of DNA replication.…”

“…The pSG5-HA-HOXD13, pSG5-HA-HOXD13IQN, and pGEX4T-HOXD13HD expression constructs were as described previously (4). Expression vectors for Myc-tagged Orc2 and Cdc6 were as described previously (58).…”

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

“…Nuclear Localization of ORC (2)(3)(4)(5)-The demonstration that either Orc1 or Orc2 protein tethered to a plasmid DNA can trigger initiation of plasmid DNA replication, whereas either Orc5 or Orc6 protein could not (41), suggests that Orc1 and Orc2 can initiate assembly of prereplication complexes, whereas Orc5 and Orc6 cannot. Mammalian ORC(2-5) can assemble spontaneously in vitro from individual ORC subunits under a variety of conditions (7)(8)(9)(10)(11)(12)(13)(14).…”