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Purification and Characterization of theSchizosaccharomyces pombe Origin Recognition Complex

2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês

10.1074/jbc.m107710200

1083-351X

Ray-Yuan Chuang, Louise Chrétien, Jianli Dai, Thomas J. Kelly,

Microtubule and mitosis dynamics

The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombeORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins. The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombeORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins. In bacteria, bacteriophage, and animal viruses the initiation of DNA replication takes place at defined nucleotide sequences known as origins of replication (1.Kornberg A. Baker T.A. DNA Replication. Freeman and Co., New York1992Google Scholar). Initiator proteins bind to such origin sequences and promote the biochemical steps leading to the establishment of replication forks. The interaction of initiator proteins with origins is less well understood in the case of eukaryotic cells where initiation of DNA replication occurs at multiple sites along chromosomal DNA (2.Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar). The best characterized eukaryotic chromosomal origins of replication are those of the budding yeastSaccharomyces cerevisiae. Like the origins of prokaryotes and animal viruses, budding yeast origins are modular in nature and are composed of several short, well defined sequence blocks distributed over a region of ∼100–150 bp (3.Campbell J.L. Newlon C.S. Broach J.R. Pringle J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1991: 41-146Google Scholar, 4.Marahrens Y. Stillman B. Science. 1992; 255: 817-823Crossref PubMed Scopus (483) Google Scholar, 5.Newlon C.S. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 873-914Google Scholar). The most highly conserved sequence block of budding yeast origins is the A domain which contains an essential 11-bp ARS consensus sequence. An additional, less well conserved sequence block, referred to as the B domain, serves to enhance the efficiency of origin utilization (3.Campbell J.L. Newlon C.S. Broach J.R. Pringle J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1991: 41-146Google Scholar, 4.Marahrens Y. Stillman B. Science. 1992; 255: 817-823Crossref PubMed Scopus (483) Google Scholar, 5.Newlon C.S. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 873-914Google Scholar). The six-subunitS. cerevisiae Origin RecognitionComplex (ScORC) 1The abbreviations used are: ScORCS. cerevisiae Origin RecognitionComplexORCorigin recognition complexHAhemagglutininARSautonomously replicating sequenceMCMminichromosome maintenance1The abbreviations used are: ScORCS. cerevisiae Origin RecognitionComplexORCorigin recognition complexHAhemagglutininARSautonomously replicating sequenceMCMminichromosome maintenance binds specifically to the ARS consensus sequence in a reaction requiring ATP (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar). Genetic and biochemical studies have established that ORC plays a central role in the initiation of DNA replication and that it functions, at least in part, to recruit essential replication factors to origins of DNA replication to form the pre-replication complex (7.Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 8.Cocker J.H. Piatti S. Santocanale C. Nasmyth K. Diffley J.F. Nature. 1996; 379: 180-182Crossref PubMed Scopus (293) Google Scholar, 9.Donovan S. Harwood J. Drury L.S. Diffley J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (429) Google Scholar, 10.Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). One such factor is Cdc6p which is required, together with ScORC, to load the MCM complex, a putative DNA helicase, onto DNA (10.Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 12.Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar) (for review, see Ref. 2.Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar).Homologues of ScORC subunits have been identified in a variety of eukaryotic species including humans (13.Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar). In addition, protein complexes containing ORC-related subunits have been identified in extracts of Xenopus laevis eggs, Drosophila melanogaster embryos, Schizosaccharomyces pombe cells, and human HeLa cells (14.Gossen M. Pak D.T. Hansen S.K. Acharya J.K. Botchan M.R. Science. 1995; 270: 1674-1677Crossref PubMed Scopus (128) Google Scholar, 15.Rowles A. Chong J.P. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 16.Moon K.Y. Kong D. Lee J.K. Raychaudhuri S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12367-12372Crossref PubMed Scopus (57) Google Scholar, 17.Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Thus, ORC has been highly conserved during evolution, suggesting the existence of common mechanisms for initiating DNA replication in all eukaryotes. However, it is not yet clear whether the interaction of ORC with origins of DNA replication in other species is similar to that in S. cerevisiae. Indeed, there is considerable evidence that the initiation of DNA replication in metazoans can occur at many sites within broad replication zones, suggesting that the sequence requirements for initiation may be more relaxed than in budding yeast (18.DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY1996: 45-86Google Scholar, 19.Dijkwel P.A. Hamlin J.L. Methods. 1999; 18: 418-431Crossref PubMed Scopus (9) Google Scholar).In previous studies we and others have shown that fission yeast origins of DNA replication differ dramatically from their budding yeast counterparts (20.Dubey D.D. Zhu J. Carlson D.L. Sharma K. Huberman J.A. EMBO J. 1994; 13: 3638-3647Crossref PubMed Scopus (106) Google Scholar, 21.Clyne R.K. Kelly T.J. EMBO J. 1995; 14: 6348-6357Crossref PubMed Scopus (107) Google Scholar, 22.Dubey D.D. Kim S.M. Todorov I.T. Huberman J.A. Curr. Biol. 1996; 6: 467-473Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Fission yeast origins have a minimal size of 500 to 1000 bp and are very rich in AT base pairs. However, they do not share a common consensus sequence comparable to the ARS consensus sequence of S. cerevisiae replication origins. In addition,S. pombe origins are characterized by a high degree of functional redundancy. Sequence blocks that are important for origin function appear to be composed of smaller AT-rich sequence elements that can be deleted individually without significantly affecting origin activity (23.Clyne R.K. Kelly T.J. Methods. 1997; 13: 221-233Crossref PubMed Scopus (18) Google Scholar). Several genetic properties of S. pombeorigins can be rationalized by the finding that the S. pombehomologue of one of the ORC subunits (SpOrc4p) contains an N-terminal DNA-binding domain consisting of nine AT-hook motifs (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar). We have suggested that binding of the N-terminal domain of SpOrc4p to appropriately spaced AT-tracts serves to tether the ORC complex toS. pombe origins of DNA replication. Consistent with this possibility, we have demonstrated that the isolated SpOrc4 subunit can bind to DNA containing a known S. pombe origin (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar). However, the DNA binding properties of the SpORC holo-complex have not been reported.To understand origin recognition and the assembly of initiation complexes in S. pombe, it will be essential to characterize the biochemical properties of SpORC. For this purpose, we have expressed all six SpORC subunits in insect cells using the baculovirus system, and we have purified the complex to near homogeneity. The purified SpORC binds with high affinity to a known origin of DNA replication in S. pombe (ars1). Our data indicate that SpORC recognizes multiple sites within ars1 DNA, consistent with the hypothesis that origin selection in S. pombe is less sequence-specific than in S. cerevisiae. The binding of SpORC to ars1 DNA is mediated by the N-terminal domain of the SpOrc4 subunit. We have demonstrated that this domain is essential for the viability of S. pombe. Finally, we have shown that SpORC interacts directly with Cdc18p, a key regulator of the initiation of DNA replication. This interaction recruits Cdc18p to origin DNA, which is a likely early step in the initiation of chromosomal DNA replication. Thus, the expression and purification of SpORC should facilitate biochemical analysis of the initiation reaction.DISCUSSIONTo understand the initiation of DNA replication at the molecular level, it will be necessary to reconstitute the initiation complex with purified proteins. Toward this end we have purified recombinantS. pombe ORC and characterized two of its primary functions, origin binding and recruitment of Cdc18p. The SpORC was produced by co-expression of recombinant subunits in the baculovirus expression system. We have developed a simple affinity purification scheme to obtain SpORC from chromatin extracts of infected insect cells. This scheme relies on epitope tags placed at the N terminus of SpOrc4p where they do not interfere with SpORC function in vivo. We have obtained highly purified SpORC and demonstrated that it consists of a stable complex of six subunits analogous to ORCs of budding yeast,X. laevis, D. melanogaster, and Homo sapiens (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar,14.Gossen M. Pak D.T. Hansen S.K. Acharya J.K. Botchan M.R. Science. 1995; 270: 1674-1677Crossref PubMed Scopus (128) Google Scholar, 15.Rowles A. Chong J.P. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17.Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).Except for the well studied case of S. cerevisiae, the nature of origins of DNA replication in eukaryotic cells remains poorly understood. Previous genetic studies have shown that S. pombe origins differ significantly from those of S. cerevisiae, suggesting that there may be substantial differences in the mechanisms of origin recognition employed by the two species (20.Dubey D.D. Zhu J. Carlson D.L. Sharma K. Huberman J.A. EMBO J. 1994; 13: 3638-3647Crossref PubMed Scopus (106) Google Scholar, 21.Clyne R.K. Kelly T.J. EMBO J. 1995; 14: 6348-6357Crossref PubMed Scopus (107) Google Scholar, 37.Dubendorff J.W. Studier F.W. J. Mol. Biol. 1991; 219: 45-59Crossref PubMed Scopus (338) Google Scholar). S. pombe origins are large, highly AT-rich sequences composed of functionally redundant elements. Importantly, they lack a common consensus sequence analogous to the ARS consensus sequence element recognized by ScORC. These properties have raised the question of whether origin recognition in fission yeast is less specific than in budding yeast. Our studies of the interaction of purified SpORC strongly suggest that this is the case. The binding of SpORC to ars1 DNA is governed by the SpOrc4 subunit, as demonstrated by the fact that the binding of the isolated SpOrc4 subunit is indistinguishable from that of the complete SpORC holo-complex. We have previously shown that the N-terminal domain of SpOrc4p, which contains nine AT-hook motifs, is necessary and sufficient for DNA binding by the isolated subunit (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar), and the present study has shown that this domain is essential for viability of fission yeast. The binding of SpORC to ars1 origin DNA appears to have a relatively low level of sequence specificity. The protein is capable of binding to multiple sites within ars1DNA with similar affinity. Although the protein binds with the highest affinity to AT-rich DNA, we also observed detectable binding to a control DNA with significantly lower AT content than that generally found in S. pombe origins. Our data are consistent with a model in which SpORC is targeted preferentially to long AT-rich regions within the S. pombe chromosomal DNA without regard to the specific nucleotide sequence of such regions. We expect that such regions would contain multiple and potentially overlapping binding sites for SpORC as in the case of ars1. One consequence of this model is that SpORC would be expected to be targeted preferentially to non-coding regions of the genome which have a higher average AT content than coding sequences. Obviously, our data do not rule out the possibility that the pattern of SpORC binding and initiation of DNA replication in vivo may be modulated by other factors such as chromatin organization and local transcriptional activity (38.Gomez M. Antequera F. EMBO J. 1999; 18: 5683-5690Crossref PubMed Scopus (69) Google Scholar). Nevertheless, it is likely that origin recognition inS. pombe is significantly less constrained by primary nucleotide sequence than in S. cerevisiae. It is possible that the same is true for metazoans, but further work will be required to assess this possibility (18.DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY1996: 45-86Google Scholar, 19.Dijkwel P.A. Hamlin J.L. Methods. 1999; 18: 418-431Crossref PubMed Scopus (9) Google Scholar).It has been demonstrated that binding of ScORC to origin DNA is strictly dependent upon binding of ATP to Orc1, and it seems likely that ATP binding and hydrolysis plays an important role in the assembly and function of the initiation complex (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar, 39.Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The binding of SpORC toars1 DNA does not require ATP, but it is possible that there are additional interactions between SpORC and DNA that are dependent on ATP. Such interactions could be difficult to detect in the presence of the strong ATP-independent binding mediated by SpOrc4p, especially if they are weak or transient in nature. One function of the N-terminal domain of SpOrc4p may be to facilitate additional protein-DNA interactions by tethering the complex to origins, thereby increasing its local concentration in the vicinity of the DNA. While such interactions may not directly contribute to origin selection, they may be critical for the downstream steps in initiation of DNA replication. This possibility is currently under investigation.Cdc6p/Cdc18p plays a key role in the initiation of S. pombeDNA replication and appears to be essential for the loading of the putative MCM helicase at origins (40.Kearsey S.E. Montgomery S. Labib K. Lindner K. EMBO J. 2000; 19: 1681-1690Crossref PubMed Scopus (90) Google Scholar, 41.Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (366) Google Scholar). It has been assumed that Cdc18p is targeted to origins via interactions with ORC, but there have been few biochemical studies to probe this interaction directly.In vitro studies with X. laevis egg extracts have shown that ORC is required for the association ofXenopus Cdc6p with chromatin (7.Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). More recently, biochemical studies with purified S. cerevisiae proteins have demonstrated interactions between Cdc6p and ScORC that alter the conformation of ScORC and modulate its DNA binding properties (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). Our data indicate that purified S. pombe ORC interacts specifically and efficiently with Cdc18p in the presence or absence of origin DNA. This finding differs from the results of studies inS. cerevisiae demonstrating that the Cdc6p-ScORC interaction is stabilized by the presence of origin DNA (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). This difference could be due to species variation or may simply reflect differences in experimental conditions, since special washing conditions were required to observe origin dependence of the Cdc6p-ORC interaction in S. cerevisiae, and the dependence was not observed in other studies (34.Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 36.Wang B. Feng L. Hu Y. Huang S.H. Reynolds C.P. Wu L. Jong A.Y. J. Biol. Chem. 1999; 274: 8291-8298Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Neither the binary SpORC-Cdc18p complex nor the ternary SpORC-Cdc18p-ars1 complex requires ATP for formation. As noted above, the possible role of ATP in the interaction betweenS. cerevisiae Cdc6p and ScORC is not yet clear, although it has been demonstrated that mutations in the nucleotide-binding motifs of Cdc6p do not abolish the interaction in vitro (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar).It appears that Cdc18p has some affinity for ars1 DNA in the absence of SpORC. This observation may be related to the recent finding that S. cerevisiae Cdc6p has an intrinsic nonspecific DNA binding activity (42.Feng L. Wang B. Driscoll B. Jong A. Mol. Biol. Cell. 2000; 11: 1673-1685Crossref PubMed Scopus (16) Google Scholar), but further work will be required to assess this possibility, since we cannot yet completely rule out the presence of a contaminating DNA-binding protein in our purified Cdc18p. In any case, the association of Cdc18p with ars1 DNA is greatly enhanced by the presence of SpORC, consistent with the hypothesis that ORC functions to recruit Cdc18p to origins via direct interactions. The ability to form ternary complexes of SpORC, Cdc18p, and ars1 in vitro should facilitate the further biochemical analysis of the assembly of initiation complexes. In bacteria, bacteriophage, and animal viruses the initiation of DNA replication takes place at defined nucleotide sequences known as origins of replication (1.Kornberg A. Baker T.A. DNA Replication. Freeman and Co., New York1992Google Scholar). Initiator proteins bind to such origin sequences and promote the biochemical steps leading to the establishment of replication forks. The interaction of initiator proteins with origins is less well understood in the case of eukaryotic cells where initiation of DNA replication occurs at multiple sites along chromosomal DNA (2.Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar). The best characterized eukaryotic chromosomal origins of replication are those of the budding yeastSaccharomyces cerevisiae. Like the origins of prokaryotes and animal viruses, budding yeast origins are modular in nature and are composed of several short, well defined sequence blocks distributed over a region of ∼100–150 bp (3.Campbell J.L. Newlon C.S. Broach J.R. Pringle J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1991: 41-146Google Scholar, 4.Marahrens Y. Stillman B. Science. 1992; 255: 817-823Crossref PubMed Scopus (483) Google Scholar, 5.Newlon C.S. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 873-914Google Scholar). The most highly conserved sequence block of budding yeast origins is the A domain which contains an essential 11-bp ARS consensus sequence. An additional, less well conserved sequence block, referred to as the B domain, serves to enhance the efficiency of origin utilization (3.Campbell J.L. Newlon C.S. Broach J.R. Pringle J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1991: 41-146Google Scholar, 4.Marahrens Y. Stillman B. Science. 1992; 255: 817-823Crossref PubMed Scopus (483) Google Scholar, 5.Newlon C.S. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996: 873-914Google Scholar). The six-subunitS. cerevisiae Origin RecognitionComplex (ScORC) 1The abbreviations used are: ScORCS. cerevisiae Origin RecognitionComplexORCorigin recognition complexHAhemagglutininARSautonomously replicating sequenceMCMminichromosome maintenance1The abbreviations used are: ScORCS. cerevisiae Origin RecognitionComplexORCorigin recognition complexHAhemagglutininARSautonomously replicating sequenceMCMminichromosome maintenance binds specifically to the ARS consensus sequence in a reaction requiring ATP (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar). Genetic and biochemical studies have established that ORC plays a central role in the initiation of DNA replication and that it functions, at least in part, to recruit essential replication factors to origins of DNA replication to form the pre-replication complex (7.Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 8.Cocker J.H. Piatti S. Santocanale C. Nasmyth K. Diffley J.F. Nature. 1996; 379: 180-182Crossref PubMed Scopus (293) Google Scholar, 9.Donovan S. Harwood J. Drury L.S. Diffley J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5611-5616Crossref PubMed Scopus (429) Google Scholar, 10.Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). One such factor is Cdc6p which is required, together with ScORC, to load the MCM complex, a putative DNA helicase, onto DNA (10.Tanaka T. Knapp D. Nasmyth K. Cell. 1997; 90: 649-660Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 12.Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar) (for review, see Ref. 2.Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar). S. cerevisiae Origin RecognitionComplex origin recognition complex hemagglutinin autonomously replicating sequence minichromosome maintenance S. cerevisiae Origin RecognitionComplex origin recognition complex hemagglutinin autonomously replicating sequence minichromosome maintenance Homologues of ScORC subunits have been identified in a variety of eukaryotic species including humans (13.Dutta A. Bell S.P. Annu. Rev. Cell Dev. Biol. 1997; 13: 293-332Crossref PubMed Scopus (340) Google Scholar). In addition, protein complexes containing ORC-related subunits have been identified in extracts of Xenopus laevis eggs, Drosophila melanogaster embryos, Schizosaccharomyces pombe cells, and human HeLa cells (14.Gossen M. Pak D.T. Hansen S.K. Acharya J.K. Botchan M.R. Science. 1995; 270: 1674-1677Crossref PubMed Scopus (128) Google Scholar, 15.Rowles A. Chong J.P. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 16.Moon K.Y. Kong D. Lee J.K. Raychaudhuri S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12367-12372Crossref PubMed Scopus (57) Google Scholar, 17.Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Thus, ORC has been highly conserved during evolution, suggesting the existence of common mechanisms for initiating DNA replication in all eukaryotes. However, it is not yet clear whether the interaction of ORC with origins of DNA replication in other species is similar to that in S. cerevisiae. Indeed, there is considerable evidence that the initiation of DNA replication in metazoans can occur at many sites within broad replication zones, suggesting that the sequence requirements for initiation may be more relaxed than in budding yeast (18.DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY1996: 45-86Google Scholar, 19.Dijkwel P.A. Hamlin J.L. Methods. 1999; 18: 418-431Crossref PubMed Scopus (9) Google Scholar). In previous studies we and others have shown that fission yeast origins of DNA replication differ dramatically from their budding yeast counterparts (20.Dubey D.D. Zhu J. Carlson D.L. Sharma K. Huberman J.A. EMBO J. 1994; 13: 3638-3647Crossref PubMed Scopus (106) Google Scholar, 21.Clyne R.K. Kelly T.J. EMBO J. 1995; 14: 6348-6357Crossref PubMed Scopus (107) Google Scholar, 22.Dubey D.D. Kim S.M. Todorov I.T. Huberman J.A. Curr. Biol. 1996; 6: 467-473Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Fission yeast origins have a minimal size of 500 to 1000 bp and are very rich in AT base pairs. However, they do not share a common consensus sequence comparable to the ARS consensus sequence of S. cerevisiae replication origins. In addition,S. pombe origins are characterized by a high degree of functional redundancy. Sequence blocks that are important for origin function appear to be composed of smaller AT-rich sequence elements that can be deleted individually without significantly affecting origin activity (23.Clyne R.K. Kelly T.J. Methods. 1997; 13: 221-233Crossref PubMed Scopus (18) Google Scholar). Several genetic properties of S. pombeorigins can be rationalized by the finding that the S. pombehomologue of one of the ORC subunits (SpOrc4p) contains an N-terminal DNA-binding domain consisting of nine AT-hook motifs (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar). We have suggested that binding of the N-terminal domain of SpOrc4p to appropriately spaced AT-tracts serves to tether the ORC complex toS. pombe origins of DNA replication. Consistent with this possibility, we have demonstrated that the isolated SpOrc4 subunit can bind to DNA containing a known S. pombe origin (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar). However, the DNA binding properties of the SpORC holo-complex have not been reported. To understand origin recognition and the assembly of initiation complexes in S. pombe, it will be essential to characterize the biochemical properties of SpORC. For this purpose, we have expressed all six SpORC subunits in insect cells using the baculovirus system, and we have purified the complex to near homogeneity. The purified SpORC binds with high affinity to a known origin of DNA replication in S. pombe (ars1). Our data indicate that SpORC recognizes multiple sites within ars1 DNA, consistent with the hypothesis that origin selection in S. pombe is less sequence-specific than in S. cerevisiae. The binding of SpORC to ars1 DNA is mediated by the N-terminal domain of the SpOrc4 subunit. We have demonstrated that this domain is essential for the viability of S. pombe. Finally, we have shown that SpORC interacts directly with Cdc18p, a key regulator of the initiation of DNA replication. This interaction recruits Cdc18p to origin DNA, which is a likely early step in the initiation of chromosomal DNA replication. Thus, the expression and purification of SpORC should facilitate biochemical analysis of the initiation reaction. DISCUSSIONTo understand the initiation of DNA replication at the molecular level, it will be necessary to reconstitute the initiation complex with purified proteins. Toward this end we have purified recombinantS. pombe ORC and characterized two of its primary functions, origin binding and recruitment of Cdc18p. The SpORC was produced by co-expression of recombinant subunits in the baculovirus expression system. We have developed a simple affinity purification scheme to obtain SpORC from chromatin extracts of infected insect cells. This scheme relies on epitope tags placed at the N terminus of SpOrc4p where they do not interfere with SpORC function in vivo. We have obtained highly purified SpORC and demonstrated that it consists of a stable complex of six subunits analogous to ORCs of budding yeast,X. laevis, D. melanogaster, and Homo sapiens (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar,14.Gossen M. Pak D.T. Hansen S.K. Acharya J.K. Botchan M.R. Science. 1995; 270: 1674-1677Crossref PubMed Scopus (128) Google Scholar, 15.Rowles A. Chong J.P. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17.Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).Except for the well studied case of S. cerevisiae, the nature of origins of DNA replication in eukaryotic cells remains poorly understood. Previous genetic studies have shown that S. pombe origins differ significantly from those of S. cerevisiae, suggesting that there may be substantial differences in the mechanisms of origin recognition employed by the two species (20.Dubey D.D. Zhu J. Carlson D.L. Sharma K. Huberman J.A. EMBO J. 1994; 13: 3638-3647Crossref PubMed Scopus (106) Google Scholar, 21.Clyne R.K. Kelly T.J. EMBO J. 1995; 14: 6348-6357Crossref PubMed Scopus (107) Google Scholar, 37.Dubendorff J.W. Studier F.W. J. Mol. Biol. 1991; 219: 45-59Crossref PubMed Scopus (338) Google Scholar). S. pombe origins are large, highly AT-rich sequences composed of functionally redundant elements. Importantly, they lack a common consensus sequence analogous to the ARS consensus sequence element recognized by ScORC. These properties have raised the question of whether origin recognition in fission yeast is less specific than in budding yeast. Our studies of the interaction of purified SpORC strongly suggest that this is the case. The binding of SpORC to ars1 DNA is governed by the SpOrc4 subunit, as demonstrated by the fact that the binding of the isolated SpOrc4 subunit is indistinguishable from that of the complete SpORC holo-complex. We have previously shown that the N-terminal domain of SpOrc4p, which contains nine AT-hook motifs, is necessary and sufficient for DNA binding by the isolated subunit (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar), and the present study has shown that this domain is essential for viability of fission yeast. The binding of SpORC to ars1 origin DNA appears to have a relatively low level of sequence specificity. The protein is capable of binding to multiple sites within ars1DNA with similar affinity. Although the protein binds with the highest affinity to AT-rich DNA, we also observed detectable binding to a control DNA with significantly lower AT content than that generally found in S. pombe origins. Our data are consistent with a model in which SpORC is targeted preferentially to long AT-rich regions within the S. pombe chromosomal DNA without regard to the specific nucleotide sequence of such regions. We expect that such regions would contain multiple and potentially overlapping binding sites for SpORC as in the case of ars1. One consequence of this model is that SpORC would be expected to be targeted preferentially to non-coding regions of the genome which have a higher average AT content than coding sequences. Obviously, our data do not rule out the possibility that the pattern of SpORC binding and initiation of DNA replication in vivo may be modulated by other factors such as chromatin organization and local transcriptional activity (38.Gomez M. Antequera F. EMBO J. 1999; 18: 5683-5690Crossref PubMed Scopus (69) Google Scholar). Nevertheless, it is likely that origin recognition inS. pombe is significantly less constrained by primary nucleotide sequence than in S. cerevisiae. It is possible that the same is true for metazoans, but further work will be required to assess this possibility (18.DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY1996: 45-86Google Scholar, 19.Dijkwel P.A. Hamlin J.L. Methods. 1999; 18: 418-431Crossref PubMed Scopus (9) Google Scholar).It has been demonstrated that binding of ScORC to origin DNA is strictly dependent upon binding of ATP to Orc1, and it seems likely that ATP binding and hydrolysis plays an important role in the assembly and function of the initiation complex (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar, 39.Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The binding of SpORC toars1 DNA does not require ATP, but it is possible that there are additional interactions between SpORC and DNA that are dependent on ATP. Such interactions could be difficult to detect in the presence of the strong ATP-independent binding mediated by SpOrc4p, especially if they are weak or transient in nature. One function of the N-terminal domain of SpOrc4p may be to facilitate additional protein-DNA interactions by tethering the complex to origins, thereby increasing its local concentration in the vicinity of the DNA. While such interactions may not directly contribute to origin selection, they may be critical for the downstream steps in initiation of DNA replication. This possibility is currently under investigation.Cdc6p/Cdc18p plays a key role in the initiation of S. pombeDNA replication and appears to be essential for the loading of the putative MCM helicase at origins (40.Kearsey S.E. Montgomery S. Labib K. Lindner K. EMBO J. 2000; 19: 1681-1690Crossref PubMed Scopus (90) Google Scholar, 41.Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (366) Google Scholar). It has been assumed that Cdc18p is targeted to origins via interactions with ORC, but there have been few biochemical studies to probe this interaction directly.In vitro studies with X. laevis egg extracts have shown that ORC is required for the association ofXenopus Cdc6p with chromatin (7.Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). More recently, biochemical studies with purified S. cerevisiae proteins have demonstrated interactions between Cdc6p and ScORC that alter the conformation of ScORC and modulate its DNA binding properties (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). Our data indicate that purified S. pombe ORC interacts specifically and efficiently with Cdc18p in the presence or absence of origin DNA. This finding differs from the results of studies inS. cerevisiae demonstrating that the Cdc6p-ScORC interaction is stabilized by the presence of origin DNA (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). This difference could be due to species variation or may simply reflect differences in experimental conditions, since special washing conditions were required to observe origin dependence of the Cdc6p-ORC interaction in S. cerevisiae, and the dependence was not observed in other studies (34.Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 36.Wang B. Feng L. Hu Y. Huang S.H. Reynolds C.P. Wu L. Jong A.Y. J. Biol. Chem. 1999; 274: 8291-8298Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Neither the binary SpORC-Cdc18p complex nor the ternary SpORC-Cdc18p-ars1 complex requires ATP for formation. As noted above, the possible role of ATP in the interaction betweenS. cerevisiae Cdc6p and ScORC is not yet clear, although it has been demonstrated that mutations in the nucleotide-binding motifs of Cdc6p do not abolish the interaction in vitro (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar).It appears that Cdc18p has some affinity for ars1 DNA in the absence of SpORC. This observation may be related to the recent finding that S. cerevisiae Cdc6p has an intrinsic nonspecific DNA binding activity (42.Feng L. Wang B. Driscoll B. Jong A. Mol. Biol. Cell. 2000; 11: 1673-1685Crossref PubMed Scopus (16) Google Scholar), but further work will be required to assess this possibility, since we cannot yet completely rule out the presence of a contaminating DNA-binding protein in our purified Cdc18p. In any case, the association of Cdc18p with ars1 DNA is greatly enhanced by the presence of SpORC, consistent with the hypothesis that ORC functions to recruit Cdc18p to origins via direct interactions. The ability to form ternary complexes of SpORC, Cdc18p, and ars1 in vitro should facilitate the further biochemical analysis of the assembly of initiation complexes. To understand the initiation of DNA replication at the molecular level, it will be necessary to reconstitute the initiation complex with purified proteins. Toward this end we have purified recombinantS. pombe ORC and characterized two of its primary functions, origin binding and recruitment of Cdc18p. The SpORC was produced by co-expression of recombinant subunits in the baculovirus expression system. We have developed a simple affinity purification scheme to obtain SpORC from chromatin extracts of infected insect cells. This scheme relies on epitope tags placed at the N terminus of SpOrc4p where they do not interfere with SpORC function in vivo. We have obtained highly purified SpORC and demonstrated that it consists of a stable complex of six subunits analogous to ORCs of budding yeast,X. laevis, D. melanogaster, and Homo sapiens (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar,14.Gossen M. Pak D.T. Hansen S.K. Acharya J.K. Botchan M.R. Science. 1995; 270: 1674-1677Crossref PubMed Scopus (128) Google Scholar, 15.Rowles A. Chong J.P. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 17.Vashee S. Simancek P. Challberg M.D. Kelly T.J. J. Biol. Chem. 2001; 276: 26666-26673Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Except for the well studied case of S. cerevisiae, the nature of origins of DNA replication in eukaryotic cells remains poorly understood. Previous genetic studies have shown that S. pombe origins differ significantly from those of S. cerevisiae, suggesting that there may be substantial differences in the mechanisms of origin recognition employed by the two species (20.Dubey D.D. Zhu J. Carlson D.L. Sharma K. Huberman J.A. EMBO J. 1994; 13: 3638-3647Crossref PubMed Scopus (106) Google Scholar, 21.Clyne R.K. Kelly T.J. EMBO J. 1995; 14: 6348-6357Crossref PubMed Scopus (107) Google Scholar, 37.Dubendorff J.W. Studier F.W. J. Mol. Biol. 1991; 219: 45-59Crossref PubMed Scopus (338) Google Scholar). S. pombe origins are large, highly AT-rich sequences composed of functionally redundant elements. Importantly, they lack a common consensus sequence analogous to the ARS consensus sequence element recognized by ScORC. These properties have raised the question of whether origin recognition in fission yeast is less specific than in budding yeast. Our studies of the interaction of purified SpORC strongly suggest that this is the case. The binding of SpORC to ars1 DNA is governed by the SpOrc4 subunit, as demonstrated by the fact that the binding of the isolated SpOrc4 subunit is indistinguishable from that of the complete SpORC holo-complex. We have previously shown that the N-terminal domain of SpOrc4p, which contains nine AT-hook motifs, is necessary and sufficient for DNA binding by the isolated subunit (24.Chuang R. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2656-2661Crossref PubMed Scopus (187) Google Scholar), and the present study has shown that this domain is essential for viability of fission yeast. The binding of SpORC to ars1 origin DNA appears to have a relatively low level of sequence specificity. The protein is capable of binding to multiple sites within ars1DNA with similar affinity. Although the protein binds with the highest affinity to AT-rich DNA, we also observed detectable binding to a control DNA with significantly lower AT content than that generally found in S. pombe origins. Our data are consistent with a model in which SpORC is targeted preferentially to long AT-rich regions within the S. pombe chromosomal DNA without regard to the specific nucleotide sequence of such regions. We expect that such regions would contain multiple and potentially overlapping binding sites for SpORC as in the case of ars1. One consequence of this model is that SpORC would be expected to be targeted preferentially to non-coding regions of the genome which have a higher average AT content than coding sequences. Obviously, our data do not rule out the possibility that the pattern of SpORC binding and initiation of DNA replication in vivo may be modulated by other factors such as chromatin organization and local transcriptional activity (38.Gomez M. Antequera F. EMBO J. 1999; 18: 5683-5690Crossref PubMed Scopus (69) Google Scholar). Nevertheless, it is likely that origin recognition inS. pombe is significantly less constrained by primary nucleotide sequence than in S. cerevisiae. It is possible that the same is true for metazoans, but further work will be required to assess this possibility (18.DePamphilis M.L. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY1996: 45-86Google Scholar, 19.Dijkwel P.A. Hamlin J.L. Methods. 1999; 18: 418-431Crossref PubMed Scopus (9) Google Scholar). It has been demonstrated that binding of ScORC to origin DNA is strictly dependent upon binding of ATP to Orc1, and it seems likely that ATP binding and hydrolysis plays an important role in the assembly and function of the initiation complex (6.Bell S.P. Stillman B. Nature. 1992; 357: 128-134Crossref PubMed Scopus (988) Google Scholar, 39.Klemm R.D. Austin R.J. Bell S.P. Cell. 1997; 88: 493-502Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The binding of SpORC toars1 DNA does not require ATP, but it is possible that there are additional interactions between SpORC and DNA that are dependent on ATP. Such interactions could be difficult to detect in the presence of the strong ATP-independent binding mediated by SpOrc4p, especially if they are weak or transient in nature. One function of the N-terminal domain of SpOrc4p may be to facilitate additional protein-DNA interactions by tethering the complex to origins, thereby increasing its local concentration in the vicinity of the DNA. While such interactions may not directly contribute to origin selection, they may be critical for the downstream steps in initiation of DNA replication. This possibility is currently under investigation. Cdc6p/Cdc18p plays a key role in the initiation of S. pombeDNA replication and appears to be essential for the loading of the putative MCM helicase at origins (40.Kearsey S.E. Montgomery S. Labib K. Lindner K. EMBO J. 2000; 19: 1681-1690Crossref PubMed Scopus (90) Google Scholar, 41.Nishitani H. Lygerou Z. Nishimoto T. Nurse P. Nature. 2000; 404: 625-628Crossref PubMed Scopus (366) Google Scholar). It has been assumed that Cdc18p is targeted to origins via interactions with ORC, but there have been few biochemical studies to probe this interaction directly.In vitro studies with X. laevis egg extracts have shown that ORC is required for the association ofXenopus Cdc6p with chromatin (7.Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). More recently, biochemical studies with purified S. cerevisiae proteins have demonstrated interactions between Cdc6p and ScORC that alter the conformation of ScORC and modulate its DNA binding properties (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). Our data indicate that purified S. pombe ORC interacts specifically and efficiently with Cdc18p in the presence or absence of origin DNA. This finding differs from the results of studies inS. cerevisiae demonstrating that the Cdc6p-ScORC interaction is stabilized by the presence of origin DNA (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). This difference could be due to species variation or may simply reflect differences in experimental conditions, since special washing conditions were required to observe origin dependence of the Cdc6p-ORC interaction in S. cerevisiae, and the dependence was not observed in other studies (34.Liang C. Weinreich M. Stillman B. Cell. 1995; 81: 667-676Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 36.Wang B. Feng L. Hu Y. Huang S.H. Reynolds C.P. Wu L. Jong A.Y. J. Biol. Chem. 1999; 274: 8291-8298Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Neither the binary SpORC-Cdc18p complex nor the ternary SpORC-Cdc18p-ars1 complex requires ATP for formation. As noted above, the possible role of ATP in the interaction betweenS. cerevisiae Cdc6p and ScORC is not yet clear, although it has been demonstrated that mutations in the nucleotide-binding motifs of Cdc6p do not abolish the interaction in vitro (11.Mizushima T. Takahashi N. Stillman B. Genes Dev. 2000; 14: 1631-1641PubMed Google Scholar). It appears that Cdc18p has some affinity for ars1 DNA in the absence of SpORC. This observation may be related to the recent finding that S. cerevisiae Cdc6p has an intrinsic nonspecific DNA binding activity (42.Feng L. Wang B. Driscoll B. Jong A. Mol. Biol. Cell. 2000; 11: 1673-1685Crossref PubMed Scopus (16) Google Scholar), but further work will be required to assess this possibility, since we cannot yet completely rule out the presence of a contaminating DNA-binding protein in our purified Cdc18p. In any case, the association of Cdc18p with ars1 DNA is greatly enhanced by the presence of SpORC, consistent with the hypothesis that ORC functions to recruit Cdc18p to origins via direct interactions. The ability to form ternary complexes of SpORC, Cdc18p, and ars1 in vitro should facilitate the further biochemical analysis of the assembly of initiation complexes. We thank Pamela Simancek and Deborah Tien for expert technical assistance and other members of the Kelly lab for stimulating discussions. We are indebted to Dr. S. Forsburg, Dr. J. Hurwitz, and Dr. S. Waddell for providing plasmids. While this paper was in revision, two papers appeared that described various aspects of the interaction of SpORC with DNA (43.Lee J.K. Moon K.Y. Jiang Y. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13589-13594Crossref PubMed Scopus (95) Google Scholar, 44.Kong D. DePamphilis M.L. Mol. Cell. Biol. 2001; 21: 8095-8103Crossref PubMed Scopus (85) Google Scholar).