Mouse and Human Homologues of the Yeast Origin of Replication Recognition Complex Subunit ORC2 and Chromosomal Localization of the Cognate Human Gene ORC2L

Takahara, Kazuhiko; Bong, M.; Brevard, Ryan; Eddy, Roger L.; Haley, L.L.; Sait, Sheila; Shows, Thomas B.; Hoffman, Guy G.; Greenspan, Daniel S.

doi:10.1006/geno.1996.0018

Cited by 42 publications

(24 citation statements)

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“…ORC was originally identified as a six-protein complex (Orc1p to Orc6p) that specifically binds to the Saccharomyces cerevisiae origins of chromosomal DNA replication (16), and its homologues have been found in various eukaryotic species, including humans (17)(18)(19)(20)(21)(22)(23). Orc1p has complete consensus sequences of both Walker A and B motifs (presumptive ATP binding sites) (24).…”

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confidence: 99%

Kinetics of ATP Binding to the Origin Recognition Complex of Saccharomyces cerevisiae

Makise

Takenaka

Kuwae

et al. 2003

Journal of Biological Chemistry

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Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The K d values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nM, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the K d values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 M), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the K d value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.

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confidence: 99%

Kinetics of ATP Binding to the Origin Recognition Complex of Saccharomyces cerevisiae

Makise

Takenaka

Kuwae

et al. 2003

Journal of Biological Chemistry

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“…CDC6/Cdc18 is closely related in sequence to one of the subunits of ORC, ORC1, over a region that includes a putative nucleotide binding motif. Yeast ORC has been demonstrated to utilize ATP for binding to DNA and to have an ATPase activity that is modulated by binding to the origin of DNA replication (1, 12), suggesting that like DNA replication initiator proteins in Escherichia coli (DnaA) or the simian virus 40 (T antigen), ATP binding and hydrolysis by the eukaryotic initiator protein will be an important regulator of the initiation process.Although DNA sequences defining an origin of replication have not yet been identified in higher eukaryotes, two members of a putative ORC complex homologous to yeast ORC1 and ORC2 have been identified so far in mammals, both in humans and in mice (13,14), suggesting a universal mechanism of initiation of DNA replication in eukaryotes. We report here the identification of a novel member of human ORC homologous to S. cerevisiae ScORC4.…”

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confidence: 99%

Identification of HsORC4, a Member of the Human Origin of Replication Recognition Complex

Quintana

Hou

Thome

et al. 1997

Journal of Biological Chemistry

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A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. Hs-ORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.Initiation of eukaryotic DNA replication involves the controlled and simultaneous firing of numerous sites of initiation. In the budding yeast Saccharomyces cerevisiae, these sites are defined by specific sequences recognized by a multisubunit complex, the origin recognition complex (ORC) 1 (1-5). All six members of ORC identified in yeast are essential for cell viability (3, 6 -11). ORC, in its pre-replicative or in its post-replicative form, is bound to DNA throughout the cell-cycle (1, 2) and could act as a platform for the recruitment of other proteins involved in the replication machinery.One of the proteins believed to be recruited by ORC before the initiation of DNA replication is the CDC6/Cdc18 (S. cerevisiae or Schizosaccharomyces pombe) protein. CDC6/Cdc18 is closely related in sequence to one of the subunits of ORC, ORC1, over a region that includes a putative nucleotide binding motif. Yeast ORC has been demonstrated to utilize ATP for binding to DNA and to have an ATPase activity that is modulated by binding to the origin of DNA replication (1, 12), suggesting that like DNA replication initiator proteins in Escherichia coli (DnaA) or the simian virus 40 (T antigen), ATP binding and hydrolysis by the eukaryotic initiator protein will be an important regulator of the initiation process.Although DNA sequences defining an origin of replication have not yet been identified in higher eukaryotes, two members of a putative ORC complex homologous to yeast ORC1 and ORC2 have been identified so far in mammals, both in humans and in mice (13,14), suggesting a universal mechanism of initiation of DNA replication in eukaryotes. We report here the identification of a novel member of human ORC homologous to S. cerevisiae ScORC4. Cloning the gene for a third member of the human origin recognition complex is an important step toward the ultimate goal of reconstituting the entire human ORC in vitro. EXPERIMENTAL PROCEDURESCloning and Sequencing-In the Expressed Sequence Tag (EST) Data base (National Center for Biotechnology Information), the partial sequence of a mouse cDNA (AA168456) was deposited with significant homology to a portion of ScORC4 from S. cerevisiae. A BLAST search with the AA168456 sequence revealed a homologous sequence human EST W23942, which in its turn identified a mouse EST AA110785. A BLAST search with the latter identified ...

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“…The second step requires a single-stranded DNA binding protein (RPA or £ Co// SSB) and results in a highly unwound species (U form DNA). The amount of U form DNA generated in XMcml 0-depleted extracts was severely reduced compared to mock-depleted extracts ( Figure 4D, lanes 2-4 versus 5-7), and this defect was rescued by the addition of recombinant XMcmlO ( Figure 4D, lane [8][9][10]. Moreover, in the absence of XMcmlO, there was no partial supercoiling of the plasmid.…”

Section: ■Xorc2mentioning

confidence: 97%

“…Although all six human homologs of yeast S. cerevisiae ORC subunits have been reported (7)(8)(9)(10)(11)(12)(13)(14), purification of a six-protein human origin recognition complex remains elusive. Endog-* This work was supported in part by National Institutes of Health Grant CA60499 (to A. D.).…”

Section: Mcmlo Is Required For Dna Replicationmentioning

confidence: 99%

“…pBS (40 ng/jd final concentration) was incubated with mockdepleted (lanes 1-4) or Mcm10-depleted egg cytosol for 30 min (lanes 5-1 o). DNA was then isolated (lane 1) or further incubated after addition of mock-depleted NPE (lanes 2-4), XMcml 0-depleted NPE (lanes 5-7), or XMcml 0-depleted NPE containing 40 ng/nl GST-XMcm10 (lanes [8][9][10], and then isolated. Origin unwinding was monitored by the appearance of negatively supercoiled "U" form DNA on a chloroquine agarose gel.…”

Section: C Lane 4)mentioning

confidence: 99%

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Mapping Critical DNA Sequence Elements Required for Amplification of erbB2 in Breast Cancer

Machida¹,

Dutta²

2003

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and FUNDING NUMBERSDAMD17-00-1-0166 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Brigham and Women's Hospital Boston, MA 02115 E-Mail:ymachida@rics. bwh.Harvard.edu Recent studies show that 80-90% of breast carcinomas are associated with gene amplification. Each amplicon most likely has an origin of replication. In Drosophila, DmORC (Origin Recognition Complex) binds to specific DNA sequences and this binding is essential for chorion gene amplification. To study the implications of replication initiator proteins to gene amplification, we characterized two proteins required for DNA replication (ORC2 and McmlO). We created hypomorphic mutation in the ORC2 gene of a cancer cell line through homologous recombination. We found that these cells failed to support the replication of an extrachromosomal plasmid bearing the oriP replicator of Epstein Barr virus (EBV). Geminin, an inhibitor of replication initiation complex, inhibited replication of this episome. The result identifies a novel means by which to cure cancer with gene amplification where the amplicons are carried as episomes. We also report that Xenopus McmlO (XMcmlO) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcmlO at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcmlO, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcmlO performs its function after pre-RC assembly and before origin unwinding. PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U IntroductionRecent studies show that 80-90% of breast carcinomas are associated with gene amplification (1,2). Unfortunately, no effort has been put so far to study the mechanism of gene amplification from a view of DNA replication. Origins of replication are first recognized and bound by a sixsubunit origin recognition complex (ORC), which was first identified in yeast. In Drosophila, DmORC (Origin Recognition Complex) binds to specific DNA sequences and this binding is essential for chorion gene amplification. One certain way to diminish the amount of amplification is to inhibit the initiation of replication in the double minute chromosome. To study the implications of replication initiator proteins to gene amplification, we characterized two proteins required for DNA replication (ORC2 and Mem 10). We also report that inh...

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Mouse and Human Homologues of the Yeast Origin of Replication Recognition Complex Subunit ORC2 and Chromosomal Localization of the Cognate Human Gene ORC2L

Cited by 42 publications

References 0 publications

Kinetics of ATP Binding to the Origin Recognition Complex of Saccharomyces cerevisiae

Kinetics of ATP Binding to the Origin Recognition Complex of Saccharomyces cerevisiae

Identification of HsORC4, a Member of the Human Origin of Replication Recognition Complex

Mapping Critical DNA Sequence Elements Required for Amplification of erbB2 in Breast Cancer

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