The
            <i>Schizosaccharomyces pombe</i>
            origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein

Lee, Joon-Kyu; Moon, Kyeong-Yeop; Jiang, Yun; Hurwitz, Jerard

doi:10.1073/pnas.251530398

Cited by 110 publications

(118 citation statements)

References 33 publications

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“…The preferential binding of Drosophila Orc6 and ORC to synthetic AT-rich DNA is also seen in the fission yeast S. pombe ORC (10,30,33). DNA binding of the S. pombe ORC to AT-rich DNA is mediated by a unique N-terminal domain in the Orc4 subunit, which contains nine AT-hook motifs known to make minor groove contacts (11).…”

Section: Discussionmentioning

confidence: 95%

Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

Balasov

Huijbregts

Chesnokov

2007

Molecular and Cellular Biology

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The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.Eukaryotic cells duplicate their genomes with remarkable precision during the course of growth and division. This process depends on stringent regulatory molecular mechanisms that couple DNA replication and cell cycle progression. To efficiently duplicate large genomes, eukaryotes have evolved a mechanism for the initiation of DNA replication that involves multiple origins of replication (ori) along the chromosomal DNA. The utilization of such sites in multicellular organisms changes during development, and this process affects both gene expression and chromosome folding. The program of such spatial and temporal activation is not understood. Although not necessarily random, the origin site selection during early Drosophila and Xenopus development appears to be less dependent on specific DNA sequences (5, 25). In agreement with this idea, a number of studies suggest that specific replicator sequences might be dispensable (22,38,52,53). Later in development origin usage becomes more specific (26, 49) and depends on many mechanisms for selection of the initiation events. Overall, with an exception of the budding yeast Saccharomyces cerevisiae, DNA sequences that define eukaryotic and especially metazoan replication origins are poorly characterized, mainly because of a lack of definitive biochemical or genetic assays (13,17,18).The hexameric origin recognition complex (ORC) is an important component for eukaryotic DNA replication. It was originally discovered in the budding yeast S. cerevisiae, and subsequent studies both in yeast and higher eukaryotes laid the foundation for understanding the functions of this important key initiation factor. ORC binds to origin sites in an ATPdependent manner and serves as a scaffold for the assembly of other initiation factors (3). ORC also directly participates in the loading of initiation factors (6, 45). Sequence rules for ORC DNA binding appear to vary ...

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Section: Discussionmentioning

confidence: 95%

Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

Balasov

Huijbregts

Chesnokov

2007

Molecular and Cellular Biology

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“…The binding through the GAL4 DNA-binding site still allows replication initiation. The GAL4 DNA-binding domain seems analogous to the naturally occurring AT-hook domain at the N terminus of Orc4 in Schizosaccharomyces pombe that has been shown to recruit ORC to AT-rich DNA segments that specify origins in S. pombe (Chuang and Kelly 1999;Lee et al 2001). Orc4 from other species lacks this AThook domain.…”

Section: An Artificial Replication Origin Created In Vivo Genes and Devmentioning

confidence: 99%

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

Takeda¹,

Shibata²,

Parvin³

et al. 2005

Genes Dev.

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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...

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“…Many yeast ARS elements have been characterized in detail, which has allowed the discovery of many cis-acting replication proteins and regulators of DNA replication (5,6). The discovery by Bell and Stillman (7,8) that the in vivo initiation of DNA replication in S. cerevisiae requires a six subunit origin recognition complex (ORC) is a major advancement in understanding the mechanism of eukaryotic DNA replication. In the G 1 phase of the cell cycle, Cdc6p, Cdt1p, minichromosome maintenance (Mcm), and Cdc45p proteins bind sequentially to DNA to form a prereplication complex (pre-RC) (9 -11).…”

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confidence: 99%

Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex

Mitkova

Biswas-Fiss

Biswas

2005

Journal of Biological Chemistry

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We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol ␣, the catalytic subunit of pol ␦, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of pol⑀. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, ␣ and ␦, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.Studies on the replication of genomes of bacteriophages, plasmids, viruses, and prokaryotic and eukaryotic cells have established general mechanisms of DNA replication (1). The eukaryotic origins of DNA replication were first discovered in the budding yeast, Saccharomyces cerevisiae, and were named autonomously replicating sequences (ARS) 1 (2-4). Many yeast ARS elements have been characterized in detail, which has allowed the discovery of many cis-acting replication proteins and regulators of DNA replication (5, 6). The discovery by Bell and Stillman (7,8) that the in vivo initiation of DNA replication in S. cerevisiae requires a six subunit origin recognition complex (ORC) is a major advancement in understanding the mechanism of eukaryotic DNA replication. In the G 1 phase of the cell cycle, Cdc6p, Cdt1p, minichromosome maintenance (Mcm), and Cdc45p proteins bind sequentially to DNA to form a prereplication complex (pre-RC) (9 -11). At the G 1 /S boundary, S phase-specific cyclin-dependent protein kinases (Cdks) and particularly the Cdc7/Dbf4 kinase transform the pre-RC into an active replication complex (10 -12). Finally, the initiation coincides with the association of the polymerase ␣/primase (pol ␣/primase) complex to the RPA-coated unwound origins (10). S and M phase-specific Cdks block the re-binding of Mcm to the chromatin and prevent a new round of initiations until mitosis (13).The Mcm protein family that is required for DNA replication in eukaryotes consists of six proteins (Mcm 2-7) with highly conserved amino acids between the six polypeptides. All of these proteins are essential for the cell viability (13, 14). The Mcm467 proteins form a relatively stable core complex and other Mcm proteins are loosely associated to it (14 -17). It has been demonstrated that a DNA helicase activity is associated with the Mcm467 complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA unwinding enzyme (18,19). Furthermore, Mcm467 remains associated with the replication fork during the elongation step of DNA replication (20). Among a number of candidate D...

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The Schizosaccharomyces pombe origin recognition complex interacts with multiple AT-rich regions of the replication origin DNA by means of the AT-hook domains of the spOrc4 protein

Cited by 110 publications

References 33 publications

Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

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

Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex

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