Multiple Functions of the Origin Recognition Complex

Multiple Functions of the Origin Recognition Complex Igor N. Chesnokov Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine, Birmingham, Alabama

The origin recognition complex (ORC), a heteromeric six‐subunit protein, is a central component for eukaryotic DNA replication. The ORC binds to DNA at replication origin sites in an ATP‐dependent manner and serves as a scaffold for the assembly of other key initiation factors. Sequence rules for ORC–DNA binding appear to vary widely. In budding yeast the ORC recognizes specific ori elements, however, in higher eukaryotes origin site selection does not appear to depend on the specific DNA sequence. In metazoans, during cell cycle progression, one or more of the ORC subunits can be modified in such a way that ORC activity is inhibited until mitosis is complete and a nuclear membrane is assembled. In addition to its well‐documented role in the initiation of DNA replication, the ORC is also involved in other cell functions. Some of these activities directly link cell cycle progression with DNA replication, while other functions seem distinct from replication. The function of ORCs in the establishment of transcriptionally repressed regions is described for many species and may be a conserved feature common for both unicellular eukaryotes and metazoans. ORC subunits were found at centrosomes, at the cell membranes, at the cytokinesis furrows of dividing cells, as well as at the kinetochore. The exact mechanism of these localizations remains to be determined, however, latest results support the idea that ORC proteins participate in multiple aspects of the chromosome inheritance cycle. In this review, we discuss the participation of ORC proteins in various cell functions, in addition to the canonical role of ORC in initiating DNA replication. KEY WORDS: Origin recognition complex, DNA replication, Chromatin, Cytokinesis. ß 2007 Elsevier Inc.

International Review of Cytology, Vol. 256 Copyright 2007, Elsevier Inc. All rights reserved.

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0074-7696/07 $35.00 DOI: 10.1016/S0074-7696(07)56003-1

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I. Introduction Eukaryotic cells duplicate their genomes with remarkable precision during the course of growth and division. This process depends on stringent regulatory mechanisms that couple DNA replication and cell cycle progression. Eukaryotic genomes are large, ranging from 107 to more than 109 base pairs, and are organized into multiple chromosomes. To eYciently duplicate these genomes, eukaryotes have evolved a mechanism in which initiation of DNA replication takes place at multiple origins of DNA replication (Ori) along the chromosomal DNA. The utilization of such sites in multicellular organisms changes during development and this process is known to aVect both gene expression programs and chromosome folding. However, the program of such spatial and temporal activation is not well understood. Despite the fact that many of the proteins involved in DNA replication are conserved in eukaryotic cells, DNA sequences that constitute eukaryotic and especially metazoan replication origins are poorly defined, mainly because of a lack of a simple and definitive biochemical or genetic assay (Cvetic and Walter, 2005; DiZey, 2004; Gilbert, 2004). Nevertheless, in the past decade there has been a dramatic advance in our understanding of cellular DNA replication. Work in simple model systems, particularly in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as in Drosophila melanogaster and Xenopus laevis, has resulted in the identification of proteins that act at origins of DNA replication to initiate DNA synthesis. In addition, a variety of genetic and biochemical approaches have defined some of the general features of the initiation pathway. Although many molecular details are still lacking, it is known that a common set of initiation proteins assembles at replication origins in all eukaryotes and that the activities of these proteins are regulated by specific protein kinases.

II. Eukaryotic Origins and Discovery of the Origin Recognition Complex According to the replicon model proposed in 1963 (Jacob and Brenner, 1963), two components are required for the initiation of DNA replication: the Initiator and the Replicator. The Initiator in this model binds a specific DNA sequence located within a genetic element called the Replicator, which is the site of initiation of DNA replication. Numerous studies in prokaryotes, viruses, and eukaryotic cells resulted in discoveries of initiator proteins, confirming this basic model. In all cases, genome duplication involves the assembly of prereplicative complex (pre‐RC) at a specific Ori and subsequent

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activation of bidirectional replication forks under the control of initiator proteins. For example, in the Escherichia coli initiator protein, DnaA binds the replication origin, oriC, at repeated motifs. DnaA then locally unwinds the DNA in an ATP‐dependent manner, facilitates helicase loading, and organizes the assembly of polymerases, primases, and other components of the replication fork. Despite the significantly greater complexity of eukaryotic chromosomes, studies performed in S. cerevisiae indicate that its genome replicates in a similar way (Bell and Dutta, 2002; Leatherwood, 1998). However, unlike prokaryotes, which use a single replicator, eukaryotic chromosomes require multiple replication origins to ensure complete replication of their genome during S phase of the cell cycle. Replication origins in yeast S. cerevisiae have been identified as autonomously replicating sequence (ARS) elements that support the propagation of extrachromosomal plasmids. Budding yeast ARSs are
100 bp in size and share an 11‐bp A/T‐rich ARS consensus sequence (ACS), an ‘‘A element’’ that is essential for origin function as shown by mutational analyses. ARS sequences also include multiple 10‐ to 15‐bp ‘‘B‐elements’’ that contribute to origin function. These well‐defined replicators of S. cerevisiae provided critical tools to identify a conserved eukaryotic initiator that directs replication events––the origin recognition complex (ORC). The identification of the ORC in S. cerevisiae was an important advance in understanding eukaryotic DNA replication. It was identified as a factor that specifically bound to the yeast ARSs (Bell and Stillman, 1992). Yeast ORC is composed of six tightly associated protein subunits, ranging from 104 kDa (Orc1) to 50 kDa (Orc6). Since its original discovery, evidence has steadily accumulated that ORC plays a central role in the initiation of DNA replication and in the recruitment of other essential replication factors to the Ori (Bell and Dutta, 2002; Bell and Stillman 1992; Dutta and Bell, 1997). Compelling evidence for the role of the ORC as the S. cerevisiae Initiator involves three complementary types of study. First, point mutations in the ACS that eliminate ORC binding in vitro and in vivo result in a loss of replicator activity (Aparicio et al., 1997; Bell and Stillman, 1992; Li and Herskowitz, 1993). Second, mutations in the genes encoding the subunits of the ORC cause DNA replication defects and prevent the association of all other replication factors at the ARS (Bell et al., 1993; Dillin and Rine, 1997, 1998; Foss et al., 1993; Micklem et al., 1993; Santocanale and DiZey, 1996). Third, other experimental data, such as in vivo footprinting, protein–DNA cross‐linking, and chromatin precipitation studies, show that the ORC is bound to S. cerevisiae Oris throughout the cell cycle (Aparicio et al., 1997; DiZey et al., 1994; Liang and Stillman, 1997; Tanaka et al., 1997). Unlike the well‐characterized replicators of S. cerevisiae, replication origins in most eukaryotic organisms are poorly defined (Bielinsky et al., 2001;

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Cvetic and Walter, 2005; Gilbert, 2004). The most obvious diVerence between Replicons in S. cerevisiae and higher eukaryotes is at the level of size. Even in the single‐cell eukaryote, S. pombe, replicons are up to 10‐fold larger than their S. cerevisiae counterparts. Replicons derived from multicellular eukaryotes are even less well defined and only a few have been mapped in detail. To make things more complicated, in multicellular organisms the number and location of origins change during development. Striking examples of such changes in origin usage are observed during development of fruit flies D. melanogaster and frogs X. laevis. In these organisms the rapid DNA replication observed in the early embryos is mediated by tens of thousands of origins spaced by as little as 4–7 kb (Blumenthal et al., 1974). At this stage of development, a cell cycle takes just 10 min to complete. Later in development, spacing between origins increases, reducing the number of used origins by more than 10‐fold (Hyrien et al., 1995; Sasaki et al., 1999). The position of Oris in early development may be stochastic with respect to DNA sequence, and in vitro experiments using early embryonic Xenopus egg extracts indicate that there is little or no sequence specificity required for the initiation of DNA replication (Coverley and Laskey, 1994). Numerous studies have been undertaken to identify the essential elements within replicators of higher eukaryotes, however, no element with properties similar to the S. cerevisiae ACS has been identified. The ORC has been conserved throughout eukaryotic evolution. ORC subunits and/or complete ORC complexes have been identified in S. pombe and various metazoans, including D. melanogaster, X. laevis, and humans (Bell, 2002). This conservation of ORC, as well as numerous other factors required for DNA replication, strongly suggests that there must be common mechanisms for the initiation of DNA replication in all eukaryotes, despite dramatic diVerences in the structure of eukaryotic origins of DNA replication and an absence of obvious conserved sequences among them. ORC genes are essential for cell survival. Mutational analysis of ORC‐related genes in S. pombe and D. melanogaster reveals defects in DNA replication (Grallert and Nurse, 1996; Landis et al., 1997; Loupart et al., 2000; Muzi‐Falconi and Kelly, 1995; Pflumm and Botchan, 2001; Pinto et al., 1999). Disruption of the Arabidopsis Orc2 gene causes a zygotic lethal phenotype (Collinge et al., 2004). In other studies, immunodepletion experiments using either X. laevis or Drosophila replication‐competent extracts indicate an absolute requirement for ORC to initiate DNA replication (Carpenter et al., 1996; Chesnokov et al., 2003; Romanowski et al., 1996; Rowles et al., 1996). Acute depletion of ORC genes in human cells by RNA interference (RNAi) results in arrested cells (Machida et al., 2005; Prasanth et al., 2002). Although no genetic evidence indicates that ORC functions in human chromosomal DNA replication, plasmids with an Epstein–Barr virus (EBV) origin are defective for replication in cells carrying an ORC2 mutation (Dhar et al., 2001) suggesting

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that EBV uses the human replication initiation machinery, and that HsORC functions in human DNA replication. Moreover, this Orc2
/‐ cell line (containing one hypomorph Orc2 allele and one null Orc2 allele) exhibited a prolonged G1 phase. Chromosomal replication otherwise was not aVected, but the low levels of Orc2 did, however, prevent replication of an exogenous plasmid containing a single EBV origin (Dhar et al., 2001b). In this case the levels of Orc2 were high enough to support chromosome replication, but not episome replication.

III. ORC Functions in DNA Replication In all cases studied, the first step in the establishment of a multiprotein assembly called the pre‐RC is the binding of the ORC to the replication origin (Bell and Dutta, 2002; Machida et al., 2005a). After the ORC binds DNA, it recruits several additional DNA replication factors to the origin (Fig. 1). Pre‐RC assembly is initiated at the M/G1 transition of the cell cycle, maintained during G1, and governed by cyclin‐dependent kinase (CDK) activity. Upon entry into the S phase, existing pre‐RCs are activated in a characteristic temporal pattern as cells progress through the S phase. This step requires an activation of another origin‐associated kinase, Cdc7, by its regulatory subunit Dbf4 and serves as the final signal‐activating replication fork movement (Brown and Kelly, 1999; Dowell et al., 1994). By the time origins fire, high CDK activity precludes the assembly of new pre‐RCs. A checkpoint ensures that the cell will not divide until replication is complete, and high levels of CDK activity are not reversed until mitosis is nearly finished. As a result the genome will be replicated once and only once per cell cycle.

A. Origin Recognition by the ORC Building of the pre‐RC requires that the ORC is bound to the origin of DNA replication. However, even in budding yeast cells it is unlikely that the ACS consensus sequence alone is suYcient to identify an ORC‐binding site or an origin (Newlon and Theis, 1993; Wyrick et al., 2001). Origin definition is even more complicated in metazoans. Initiation sites are uniformely distributed throughout the genome and in embryonic cells many of them are used. As the embryo develops, initiation events become restricted to specific sites. Several studies were undertaken to identify origins and ORC‐binding sites across the S. cerevisiae and Drosophila genome (MacAlpine et al., 2004;

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ORC

Cdc6

M Cdt1

Cdc6

Cdt1 ORC

Late M

Geminin MCMs

Cdc6 MCMs

Cdt1 ORC

GINS

G1

Cdc45 Cdt1 Cdc6 ORC MCMs Cdc45

G1/S

MCM GINS 10 MCM 10 Cdc6 Cdt1 ORC GINS

Cdc45 MCMs MCM 10 GINS

RPA

MCM MCMs 10

S

Cdc45

ORC FIG. 1 Assembly of the prereplicative complex at replication origins (simplified mode). The binding of the ORC to the replication origin is the first step in the assembly of the pre‐RC. Beginning in late mitosis, Cdc6 protein joins origin‐bound ORC followed by the Cdt1 protein. ORC, Cdc6, and Cdt1 are required to load the six‐protein MCM complex, a replicative helicase, onto the DNA. In a late step requiring CDK activity, MCM10, Cdc45, and GINS are added to the prereplication complex.

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Raghuraman et al., 2001; Wyrick et al., 2001) by cross‐linking the ORC to its binding sites in vivo and using high‐resolution genomic microarray to determine replication timing, identify replication origins, and map protein‐ binding sites along a chromosome arm. In these studies, DNA fragments cross‐linked to the ORC were isolated by chromatin immunoprecipitation and subjected to DNA microarrays to identify the associated DNAs. It turned out that a majority of these binding sites are in the intergenic regions and more than 90% colocalize with the binding sites of the minichromosome maintenance (MCM) proteins during G1, suggesting their direct role in the formation of pre‐RCs. This approach allows the prediction of the sites of possible origins to within 1 kb, especially when combined with other methods to confirm the sites of replication initiation. The ORC was found at specific chromosomal sites, many of which coincide with early activating origins. However, more ORC‐binding sites than origins were predicted, suggesting that some of these sites might be involved in other cellular functions. It is interesting that the temporal pattern of replication also correlated with the density of active transcription on the chromosomes (MacAlpine et al., 2004). The molecular features of ORC‐binding sites include increased AT content and association with a subset of RNA Pol II‐ binding sites (MacAlpine et al., 2004). It is possible, that the distribution of transcription along the chromosome acts locally to influence origin selection and globally to regulate origin activation. In metazoans, the search for DNA sequences that define Ori sites has been hampered by the lack of convenient genetic or biochemical assays for critical cis‐acting motifs (Gilbert, 1998). To further increase the complexity, a developmental program regulates origin selection functions in temporal and tissue‐ specific ways and DNA elements spaced over large distances participate in replicator activity (Cimbora and Groudine, 2001; Cimbora et al., 2000; Lu et al., 2001). Therefore studies of metazoan ORCs have been less clear in respect to specific ORC‐binding sites. In the early embryonic stages of Drosophila and Xenopus, origin site selection does not appear to depend on the specific DNA sequence, however, it is not necessarily random (Blow et al., 2001; Hyrien et al., 2003), indicating that specific ‘‘replicator’’ sequences are dispensable (Harland and Laskey, 1980; Mechali and Kearsey, 1984; Smith and Calos, 1995; Spradling and Orr‐Weaver, 1987). Moreover, studies of DNA replication in extracts derived from embryonic cells indicate that practically any DNA sequence can be replicated (Coverley and Laskey,

Pre‐RC assembly is initiated at the M/G1 transition of the cell cycle and maintained during G1. Upon entry into S phase, existing pre‐RCs are activated by kinases in a characteristic temporal pattern. Cdc6, Cdt1, and MCMs are removed and/or inactivated during S and G2 phases to prevent second round pre‐RC formation during the cell cycle.

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1994). Specific origin usage occurs later in development, showing that some mechanism(s) for selection of initiation zones or sites must exist (Hyrien et al., 1995; Sasaki et al., 1999). However, despite this apparent lack of specificity, studies of Xenopus and Drosophila egg extracts indicate that the ORC is a necessary component for DNA replication (Blow et al., 2001; Chesnokov et al., 1999; Rowles et al., 1999). The ORC, especially in metazoan species, does not have the ability to select origins based solely on its own aYnity for specific DNA sequences. Metazoan replication origins are also determined by epigenetic information because site‐specific initiation in those species is developmentally acquired. The mechanisms by which the ORC is localized to origins of replication remain incompletely understood, however, several mechanisms have been described that could be involved in targeting the ORC to the specific sites on a DNA. First, the ORC itself has a DNA‐binding ability. Second, the association of other replication factors with ORC could enhance its interaction with origin sequences. Third, other factors, such as transcription factors, that interact specifically with DNA could be involved in recruiting the ORC to specific sites in the genome. Fourth, characteristics and state of chromatin structure in specific regions of the genome could restrict the areas in which the ORC can function. Finally, certain specific conditions may also influence ORC binding and assembly of the pre‐RC.

1. DNA Binding Activity of the ORC The best understood biochemical activity of the ORC in a wide variety of species is its ATP‐dependent DNA binding. This binding is sequence specific in yeast S. cerevisiae. In the other eukaryotic species ORC–DNA binding is significantly less specific and exact sequences that direct ORC binding are unknown. The S. cerevisiae ORC binds ARS sequences in a sequence‐specific manner (Bell and Stillman, 1992; Rao and Stillman, 1995; Rowley et al., 1995). One component of a recognition site is the 11‐bp ACS. Further studies revealed additional interactions within the adjacent B1 domain and sequences between the A and B1 region (Lee and Bell, 1997; Rao and Stillman, 1995; Rowley et al., 1995). Single base substitutions within either of these sequences reduce ORC binding in vitro and also reduce replication activity in vivo. The details of the interaction between the ORC and origin DNA are complicated and have not yet been completely worked out. Analysis of modified DNA substrates demonstrated that DNA‐bound ORC primarily interacts with the A‐rich strand of the ACS, suggesting that other replication factors can gain access to the opposite strand of the ACS without displacing the ORC (Lee and Bell, 1997). It is not yet clear which subunits of the ORC determine the specificity of

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binding. As was shown by protein–DNA cross‐linking studies in S. cerevisiae, four out of the six ORC subunits (Orc1, Orc2, Orc4, and Orc5) were closely associated with the origin DNA (Lee and Bell, 1997). Only the Orc6 subunit is dispensable for sequence‐specific DNA binding, although it is still required for ORC function in vivo (Lee and Bell, 1997). In contrast, studies of Drosophila ORC indicate that the Orc6 subunit is required for eYcient DNA binding as well as for in vitro DNA replication (Chesnokov et al., 2001). Structural and biochemical studies in recent years begin to clarify the complex nature of the ORC–DNA interface by determining the DNA‐ binding motifs of the ORC involved in origin recognition (Chastain et al., 2004; Speck et al., 2005). The sequence comparison had not previously identified any clear DNA‐binding motifs in any ORC subunit from any species. The determination of the crystal structure of an archaeal Cdc6, which is closely related to the Orc1 subunit, has revealed a candidate DNA‐binding domain (Liu et al., 2000), a region of the C‐terminus that forms a fold related to the winged‐helix DNA‐binding domain. This motif is conserved in both Cdc6 and Orc1 proteins derived from multiple species, suggesting that this region of Orc1 mediates, at least in part, DNA binding by ORC. It has been revealed that budding yeast ORC subunits from 1 to 5 all have sequences and structure typical for AAAþ fold (Speck et al., 2005). Moreover, electron microscopic reconstruction revealed the structure of S. cerevisiae ORC with dimensions consistent with the DNA‐binding studies (Speck et al., 2005). Replicons in the fission yeast S. pombe appear to consist of largely asymmetric stretches of AT that do not show consensus sequence elements. DNA binding by the ORC in this organism is dependent upon a special N‐terminal domain of the Orc4 subunit that contains multiple A/T hook motifs that can each recognize such DNA elements. S. pombe Orc4 can bind to origin DNA even in the absence of other ORC subunits (Chuang and Kelly, 1999). This motif is absent in Orc4 homologs from any other species. The ‘‘AT hook’’ motifs are found in a number of DNA‐binding proteins, including the HMG‐ I(Y) family of mammalian chromosomal proteins (Bustin and Reeves, 1996). The AT hook motif is known to bind to the minor groove of AT tracts (Bustin and Reeves, 1996). Interestingly, studies of other AT hook proteins suggest that they can recognize or induce structural changes in bound DNA. In vitro binding experiments using S. pombe ORC have have shown that the protein can interact with both the ars3002 and ars1 origins of replication (Chuang and Kelly, 1999; Kong and DePamphilis, 2001; Lee et al., 2001; Moon et al., 1999) and also have shown the importance of several specific AT‐rich sites within these origins (Kong and DePamphilis, 2001; Lee et al., 2001). Other S. pombe ORC subunits did not have any sequence‐specific DNA‐binding activity on their own, nor did they alter the interaction of pre‐bound Orc4 with the origin DNA (Kong and DePamphilis, 2001; Lee

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et al., 2001), indicating that the Orc4 subunit is largely responsible for the sequence specificity of the ORC in fission yeast. The N‐terminal domain of S. pombe Orc4 may function to tether the ORC complex to origins of DNA replication. This interaction is independent of ATP. However the tethered complex may also make ATP‐dependent contacts with additional sites in the origin to nucleate formation of the initiation complex (Chuang and Kelly, 1999). Metazoan origins are much larger than the small origins of budding yeast (Aladjem et al., 1998; Gilbert, 1998) and similar in size and complexity to those in S. pombe. The identification and subsequent reconstitution of the Drosophila ORCs have made it possible to explore ORC–DNA interactions in a metazoan model system (Austin et al., 1999; Chesnokov et al., 1999, 2001; Gossen et al., 1995). Immunostaining of Drosophila cells with an antibody raised against the Drosophila Orc2 subunit indicated that the protein is distributed to many chromosomal loci, which may represent potential replication origins (Pak et al., 1997). Interestingly, Drosophila ORC in those experiments showed a remarkable preference for heterochromatic regions suggesting that it may also play a role in chromatin organization in addition to its role in DNA replication (Pak et al., 1997). Drosophila also exhibits a specialized DNA replication reaction that amplifies two clusters of chorion genes in ovarian follicle cells surrounding the developing oocyte during a specific stage in oogenesis (Spradling, 1999). This system became one of the most tractable systems for studying origin choice and ORC localization in metazoans (Calvi and Spradling, 1999). In these somatic cells, the chorion genes on the third and X chromosomes undergo site‐specific DNA amplification to allow for a rapid increase in the number of templates for later transcription of the egg shell genes. Chorion gene amplification requires ORC as well as specific cis‐regulatory elements ACE3 (Amplification Control Element 3) and orib (Calvi et al., 1998; Landis et al., 1997; Orr‐Weaver, 1991). ACE3 is
400 bp in size and contains several partially redundant sequence elements that contribute to its function (Orr‐Weaver et al., 1989). Immunofluorescence studies show that Drosophila ORC is localized to the region of chorion amplification in follicle cell nuclei during Drosophila egg development (Royzman et al., 1999). Moreover, the ACE3 element alone is suYcient to drive this localization (Austin et al., 1999). Chromatin immunoprecipitation studies indicate that ACE3 can target Drosophila ORC not only to sites within ACE3 elements itself but also to sites within adjacent DNA sequences (Austin et al., 1999). In addition to these studies of the in vivo localization of Drosophila ORC, in vitro DNA‐binding studies have been performed with ORC purified from Drosophila embryos or reconstituted from recombinant proteins (Austin et al., 1999; Chesnokov et al., 1999, 2001; Remas et al., 2004). Purified ORC in these studies bound ACE3 and orib DNA in an ATP‐dependent

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manner but with little specificity (Austin et al., 1999; Chesnokov et al., 2001; Remas et al., 2004). Moreover, the ORC from Drosophila displays at best 6‐ fold diVerences in the relative aYnities to DNA from the third chorion locus versus random fragments in vitro. It appears that the intrinsic DNA‐binding specificity of the ORC is not suYcient to target the ORC to origins of replication in vivo. The chemical probing and DNase I protection experiments did not identify a discrete binding site for the ORC on any of these fragments (Remus et al., 2004). On the other hand, the topological state of the DNA significantly influences the aYnity of the ORC to DNA (Remus et al., 2004). It is found to be 30‐fold higher for negatively supercoiled DNA as compared to relaxed or linear DNA, indicating that origin specification in metazoa likely involves mechanisms other than simple replicator–initiator interactions and that in vivo other proteins must determine ORC’s localization (see further discussion). Interestingly, the same recombinant Drosophila ORC specifically binds the amplification origin II/9A of the fly Sciara coprophila at a sequence near the site of initial nucleotide incorporation (Bielinsky et al., 2001). This same region is also associated with Sciara ORC in vivo. In all cases observed DNA binding by the ORC is ATP dependent. A number of questions remain unresolved, particularly the issue of whether the Drosophila ORC–ACE3 interaction is relevant to the interaction of the ORC with normal origins. During amplification of the chorion gene clusters, other cellular origins remain silent. Most likely, Drosophila ORC is targeted to normal origins by a diVerent mechanism, involving additional chromatin proteins or replication factors or possibly some unknown aspects of chromatin organization. Attempts to identify stable autonomously replicating sequences in human cells have been largely unsuccessful. Various studies have mapped
20 mammalian origins of bidirectional replication using a variety of approaches including 2D gels and nascent strand abundance assays (Gilbert, 2001). Some of these origins, such as the human b‐globin locus, localize to a relatively discrete chromosomal location. The second group of origins consists of large zones of initiation, 10‐ to 50‐kb regions in which replication begins from multiple dispersed sites. The DHFR locus in Chinese hamster ovary cell is a typical example of this group. Despite the discovery of these mammalian origins, no consensus DNA sequence emerges among them. Reconstituted, highly purified human ORC has DNA‐binding ability, which is stimulated by ATP (Vashee et al., 2003). Human ORC binds preferentially to AT‐rich sequences, but does not eVectively discriminate between natural DNA fragments that contain known human origins and control fragments. Complex restored DNA replication in Orc2‐depleted Xenopus extracts suggests that it is functional. Recombinant human ORC largely consisting of subunits 1–5 stimulates initiation from any DNA sequence (Vashee et al., 2003), suggesting that in metazoans, initiation of DNA replication may occur in a seemingly random pattern.

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It appears that mammalian and Drosophila ORCs, the presumptive initiators, do not have a strong preference for binding to particular DNA sequences but favor AT‐rich DNA (Remus et al., 2004; Schaarschmidt et al., 2004; Vashee et al., 2003). Instead, Drosophila ORC prefers negatively supercoiled DNA, with the DNA topology proving to be an important determinant for ORC binding (Remus et al., 2004). It appears that origin specification involves mechanisms other than simple recognition of DNA sequence by the ORC in metazoans. Overall, these studies suggest that ORCs isolated from metazoan species retain some DNA sequence preferences, but apparently not at the level observed for ORCs derived from S. cerevisiae. Other factors must therefore play an important role for targeting the ORC to replicators in vivo.

2. The Role of Replication Factors in ORC Binding to the Origins The replication factors such as Cdc6 may also contribute to the origin recognition by ORC. Binding of Cdc6 to the ORC is a key step in the assembly of a pre‐RC. Mizushima and coworkers (2000) reported that the association between budding yeast ORC and Cdc6 protein was facilitated by origin‐ containing DNA. They observed that this interaction also reduced the oV‐ rate of the ORC from origin but not from nonorigin fragments. An intact Cdc6p ATP‐binding motif was required for the reduced oV‐rate. In addition, the interaction between the ORC and Cdc6p appeared to alter the conformation of one or more ORC subunits and this change could lead to enhanced interaction with the origin (Mizushima et al., 2000). S. cerevisiae Cdc6 binds cooperatively with the ORC on DNA in an ATP‐dependent manner (Speck et al., 2005). This binding induces a change in the pattern of origin binding that requires the Orc1 ATPase. Specific origin mutations were identified that did not interfere with the interaction between the ORC and DNA, but blocked the interaction with Cdc6. It is interesting that the ORC–Cdc6 complex formed a ring‐shaped structure similar to the structure of MCM helicase. This structure also is predicted to contain six AAAþ polypeptides, analogous to other ATP‐dependent protein complexes (Speck et al., 2005). Cdc6 may also increase the DNA‐binding specificity of the ORC from metazoan species, where DNA recognition by the ORC alone has little DNA sequence specificity. Indeed, Cdc6‐induced stabilization of ORC binding to sperm chromatin has been observed in Xenopus crude egg extracts in the presence of ATPgS (Harvey and Newport, 2003b). It is possible that in metazoan species, the ORC–Cdc6 complex determines the location of origins of DNA replication, most likely in conjunction with other DNA‐binding proteins.

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3. The Role of Transcription and Transcription Factors in Targeting the ORC to the Origins Transcription itself may also influence ORC localization. There is a strong tendency for origins and ORC‐binding sites to be located in intergenic regions (Brewer, 1994; Gilbert, 2001; MacAlpine et al., 2004; Wyrick et al., 2001). Studies in S. cerevisiae and Drosophila have shown that origins and ORC‐binding sites appear to be restricted to intergenic regions even in poorly transcribed regions of the genome. It is also very likely that the open chromatin structure associated with the control regions of genes may provide increased access for the ORC or other replication factors. Evidence for molecular mechanisms that connect transcription and replication has been described. In yeast, the B3 element close to ARS1 is the binding site for the transcription factor Abf1 and can be substituted by the other transcription factor binding sites (Marahrens and Stillman, 1992). In fission yeast, active origins lie close to promoter regions (Gomez and Antequera, 1999). In the metazoans Drosophila and Xenopus, initiation of DNA replication is regulated developmentally. After the mid‐blastula transition (when transcription begins) replication switches from a random localization to a preferential localization in the promoter region (Hyrien et al., 1995; Sasaki et al., 1999). Studies of DNA replication at the Drosophila chorion gene amplification locus suggested a role for DNA‐binding factors in targeting the ORC to specific genomic loci. For instance, it was shown that the Drosophila analogs of the E2F1 transcription factor (dE2F1) and Rb (Rbf) are associated with a subset of ORC in Drosophila cells (Bosco et al., 2001; Royzman et al., 1999). Even more importantly, mutant alleles of dE2F1, Rbf, and the Drosophila Dp analog have strong eVects on the localization of the ORC and the function of the ACE3 amplification origin (Bosco et al., 2001; Royzman et al., 1999). The sequence elements localized within ACE3 and ori‐b are also recognized by a five‐subunit complex containing a Drosophila homolog of the Myb protooncogene (Beall et al., 2002). This complex was originally identified as an activity present in Drosophila extracts that specifically recognizes two critical control elements (ACE‐3 and ori‐b) required for chorion gene DNA replication‐mediated amplification in the follicle cells surrounding the developing oocyte (Beall et al., 2002; Orr‐Weaver et al., 1989). Mutations in the Myb‐binding site in ACE3‐containing transgenes result in reduced amplification of such reporters. Moreover, somatic follicle cell clones devoid of Myb are defective for chorion gene amplification (Beall et al., 2002). Further studies revealed that the Myb complex may serve dual functions in both the activation and repression of DNA replication (Beall et al., 2004).

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This function may depend upon the presence or absence of other factors at a given chromosomal location and/or developmental context. In early Xenopus development, transcription is repressed and DNA replication initiates at nonspecific sites. It has been shown that a site‐specific DNA replication origin can be induced in this context by the assembly of a transcription domain (Danis et al., 2004). Deletion of the promoter element abolishes site‐ specific initiation, and its relocalization to an ectopic site induces a new origin of replication. Danis et al. (2004) did not detect any change in the replication rate, but observed a restriction of the origin by the transcription activator. In human cells, the viral transcription factor EBNA1 plays an important role in targeting human ORC to the EBV replication origin (Chaudhuri et al., 2001; Dhar et al., 2001b; Schepers et al., 2001). If a sequence‐specific DNA‐ binding factor like EBNA1 helps recruit the ORC to oriP, recruitment of the ORC to cellular chromosomal origins of replication might also be dependent on sequence‐specific DNA‐binding factors. Such indirect recruitment of mammalian ORC to chromatin by diverse sequence‐specific factors might explain why it has been so diYcult to find a single DNA sequence that acts as a replicator in mammalian chromosomal origins of replication. Otherwise, each origin would contain a consensus‐binding site for the ORC recruiting factor. It is possible that transcription enhancers perform similar functions in transcription and DNA replication, but through diVerent mechanisms. Transcription enhancers contribute to chromatin opening, and therefore activate transcription. However, transcriptional enhancers do not specify the sites of transcription initiation. At the same time, their role in DNA replication might not be to activate replication, but to contribute to site specificity. This becomes important later in development, when maternal replication initiation proteins become limiting, and chromatin becomes less accessible, resulting in reducing nonspecific initiation of DNA replication.

4. The Role of Chromatin Structure in Origin Specification In vivo studies in yeast indicate that positioning a nucleosome over the ORC‐ binding site at an origin inactivates origin function (Simpson, 1990) and even subtle changes in the local chromatin structure have a significant impact on origin usage; however, this eVect may be more complex and go beyond the level of ORC–DNA binding (Lipford and Bell, 2001). This also may explain the increased restriction of origin sites as developing embryonic cells begin transcription (Hyrien et al., 1995; Sasaki et al., 1999). Chromatin structure around a promoter modulates the transcriptional activity of genes. Activators and suppressors of transcription often remodel chromatin by modifying or repositioning histones. Activity of diVerent

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origins can also change during development due to epigenetic influences. Such developmental specificity occurs in Drosophila, where origins that control amplification of the eggshell (chorion) protein genes are amplified specifically during a defined stage of somatic follicle cell cycle. It was shown that chromatin acetylation is critical for this developmental transition in origin specificity (Aggarwal and Calvi, 2004). Histones around ORC‐binding sites are hyperacetylated during gene amplification in Drosophila follicle cells (Aggarwal and Calvi, 2004). Mutation of the histone deacetylase (HDAC) Rpd3 induced genome‐wide hyperacetylation, genomic replication, and a redistribution of the ORC in amplification‐stage cells. This was independent of eVects on transcription. Tethering histone acetyltransferase (HAT) and HDAC to origins resulted in an increase or decrease of origin activity, respectively (Aggarwal and Calvi, 2004). In Xenopus, chromatin immunoprecipitation experiments suggest that site‐ specific acetylation of histones favors the Initiator function of active origin selection (Danis et al., 2004). It again appears that the specification of active DNA replication origins is governed by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes. One of the best candidates for the HAT that might work at origins is HBO1 (histone acetyltransferase binding to ORC). HBO1 belongs to a MYST family histone acetyltransferase and associates with pre‐RC components such as Mcm2 and Orc1 (Burke et al., 2001; Iizuka and Stillman, 1999). In addition, another histone acetyltransferase of the MYST family, Sas2, has been genetically linked to the ORC, although it is not clear yet whether it plays a role in regulating replication initiation (Ehrenhofer‐Murray et al., 1997). Interaction of HAT with pre‐RC components suggests that histone acetylation around replicons might be an active process in which chromatin is remodeled by Initiators. Another possibility is that replication factors likely have easier access to DNA in open chromatin around transcriptionally active genes. Supporting this model, the formation of active transcription complexes on chromatin can specify an origin on a plasmid in Xenopus extracts (Danis et al., 2004). This process does not require active transcription, but the histones around the transcription complex are acetylated, suggesting that changes in chromatin structure by transcription complexes nucleate origins. Interestingly, origin specification was not at the level of ORC binding, because the ORC was not enriched at origins in this experiment. Thus, origin specification by histone acetylation might occur after ORC binding to the chromatin, but before the origin decision point at the G1 phase of the cell cycle, the time at which origins become specific (Okuno et al., 2001). Other epigenetic parameters that may also contribute to origin specification include nucleotide pool levels and DNA methylation. Using molecular combing, Anglana and coworkers (2003) identified five secondary

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(low‐frequency) initiation sites within 128 kb of the single, primary (high‐ frequency) oriGNA13 previously identified by them in Chinese hamster cells. Remarkably, their results reveal that initiation at the primary origin represses initiation at the secondary origins. Moreover, increasing the nucleotide pool by addition of DNA precursors to the culture of cells reduces the frequency of initiation at secondary origins, whereas reducing the nucleotide pool by addition of hydroxyurea (a specific inhibitor of ribonucleotide reductase) increases the frequency of initiation at secondary origins. Cells reversibly respond to a reduction in nucleotide availability by slowing the rate of replication fork progression; in addition, the eYciency of initiation at ori GNAI3 is lowered while other normally dormant origins in the region are activated, which results in an overall increase in the density of initiation events. Thus, nucleotide pools are involved in the specification of active origins, which in turn defines their density along chromosomes. Anglana et al. (2003) suggest that the eYciency of some origins relies more on nucleotide availability and/or fork progression rate than on specific cis‐sequences. It has been reported (Harvey and Newport, 2003a) that DNA replication can be targeted by default to a low‐methylation DNA piece inserted in a fully methylated plasmid. In a Xenopus egg replication system, the ORC does not bind to CpG methylated DNA resulting in the inhibition of DNA replication. Insertion of low‐density CpG DNA of at least 1.2 kb into methylated plasmids rescues both ORC binding and DNA replication. ORC binding, however, was restricted to low‐CpG‐density DNA. On the other hand, MCM proteins were loaded onto both weakly and highly methylated DNA and occupied approximately 2 kb of DNA. In this case origin specification will occur mainly by decreasing initiation events at sites distant from an initiation zone around the low‐methylated region. The rate of DNA replication would not change, but a specific origin would become prominent because initiation events previously occurring at the other sites would carry over into the chosen origin. Concurrent with these results, in mammalian cells it was found that at some loci, DNA methylation either directly or indirectly determines where replication begins (Rein et al., 1999).

B. ATPase Activity of the ORC The formation of pre‐RC is regulated by ATP. In all species the ORC requires ATP to interact with origin DNA (with an exception of fission yeast ORC where initial binding is facilitated in an ATP‐independent way by the AT hook domain of the Orc4 subunit). Three subunits of the ORC (Orc1, Orc4, and Orc5) contain potential ATP‐binding sites based on amino acid sequence analysis. Studies in budding yeast have shown that mutations in ATP‐binding motifs of Orc1 have a lethal eVect (Klemm et al., 1997).

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Similar mutations in Drosophila and human Orc1 proteins resulted in an ORC complex that was unable to bind ATP, to bind DNA, and to support DNA replication in vitro (Chesnokov et al., 2001; Giordano‐Coltart et al., 2005). Mutations in the ATP‐binding motifs of Orc4 and 5 did not have obvious phenotypes for budding yeast, Drosophila, and human ORC (Chesnokov et al., 2001; Giordano‐Coltart et al., 2005; Klemm et al., 1997) suggesting that ATP binding to these domains might be required for nonreplicative ORC functions, such as DNA replication checkpoints or transcriptional silencing. Mutagenesis studies indicate that ATP binding by the ORC, but not hydrolysis of ATP, is required for DNA binding by the ORC (Chesnokov et al., 2001; Giordano‐Coltart et al., 2005; Klemm et al., 1997). In all cases only mutations in the Orc1 subunit ATP‐binding motif interfere with ATP hydrolysis. Given that a nonhydrolyzable ATP analog inhibits the loading of MCM2‐7, but not the loading of the ORC and Cdc6 (Gillespie et al., 2001; Harvey and Newport, 2003b), it seemed likely that ATP hydrolysis is used to load MCM2‐7 on chromatin. Indeed, it proved to be the case when the mechanism of ATP hydrolysis by yeast ORC was studied in more details. It was shown that ATP hydrolysis by the ORC requires the coordinate function of Orc1 and Orc4 subunits (Bowers et al., 2004). Critical arginine involved in the formation of a putative arginine finger in the Orc4 subunit is required for Orc1 ATP hydrolysis, but not ATP or DNA binding. Preventing ORC‐dependent ATP hydrolysis inhibits reiterative loading of a helicase Mcm2‐7 during pre‐RC formation (Bowers et al., 2004). In the emerging model, the bound ATP is required for stable DNA binding by the ORC and it is not immediately hydrolyzed. Instead, hydrolysis of this ATP happens during a subsequent step in the replication process––the repeated loading of Mcm2‐7 complexes. This event requires coordinated and sequential ATP hydrolysis by both ORC and Cdc6 (Bowers et al., 2004; Randel et al., 2006). The ORC, therefore, emerges as a part of a helicase loading machinery that facilitates the assembly of multiple Mcm2‐7 complexes that play a crucial role in origin function. It also appears that ATP hydrolysis by the ORC and/or Cdc6 causes a conformational change resulting in the release of helicase and allows additional molecules of Mcm2‐7 to be loaded (Bowers et al., 2004; Randel et al., 2006). The inhibitory eVect of DNA fragments containing origin sequences on ATP hydrolysis by budding yeast ORC was also observed. ATPase activity of Drosophila ORC was inhibited by any added DNA. Budding yeast ORC also has a high aYnity for single‐stranded DNA (ssDNA) (Lee et al., 2000), which is neither sequence specific nor ATP dependent. The binding of the ORC to ssDNA stimulates the rate of ATP hydrolysis up to a 20‐fold increase relative to ORC bound to origin DNA. Moreover, electron microscopy analysis of ORC function indicates that binding to ssDNA stabilizes an altered structure. Because ssDNA is a direct consequence of the

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initiation process after unwinding of the DNA, these observations suggest that the generation of ssDNA during initiation is coupled to activation of the ORC ATPase and alteration of its structure. The ssDNA‐stimulated activation of the ORC ATPase could also contribute to the loading of the MCM complex around ssDNA at the time of initiation. In this case, the ssDNA stimulation of ORC ATPase activity would resemble the DNA stimulation of ATP hydrolysis of the clamp loader (Lee et al., 2000). In humans, it was shown that ATP is required for maintenance of ORC integrity and specific subunit interactions within ORC and that it facilitates whole complex formation (Ranjan and Gossen, 2006). The human Orc4 subunit plays a critical role in this process, suggesting that besides its previously identified role in DNA binding, ATP also serves as a structural cofactor for human ORC (Ranjan and Gossen, 2006).

C. Assembly of the Prereplicative Complex Once bound to DNA the ORC plays a critical role in the recruitment of additional replication factors to the origin and formation of the pre‐RC (Bell, 2002; Bell and Dutta, 2002; DiZey, 2004; Machida et al., 2005a). The recruitment of Cdc6 and Cdt1 occurs first, followed by Mcm2‐7, a hexameric helicase complex responsible for unwinding parental DNA strands. Although ORC, Cdc6, and Cdt1 are required to load MCM2‐7 helicases onto chromatin, the exact mechanism of action of these proteins is poorly understood. The ring‐shaped structure of MCM2‐7 encircles the DNA, suggesting that ORC and Cdc6 might act as an ATP‐dependent clamp‐ loader analogous to the RF‐C clamp‐loader for the proliferating cell nuclear antigen (PCNA) (Mendez and Stillman, 2003; Speck et al., 2005a). As cells continue through the G1 phase toward S phase transition additional replication factors are recruited to the origin including cyclin‐dependent and Dbf4‐ dependent kinases (CDKs and DDK), Cdc45, Mcm10, GINS complex, the three eukaryotic DNA polymerases, and the eukaryotic ssDNA‐binding protein, RPA (Fig. 1) (Bell and Dutta, 2002; DiZey, 2004; Machida et al., 2005a). In the absence of the ORC the assembly of the pre‐RC fails in both cells and cell‐free systems; even so the ORC by itself has been shown to interact directly with only a small subset of these factors. Documented interactions include Cdc6 (Klemm and Bell, 2001; Mizushima et al., 2000; Saha et al., 1998; Seki and DiZey, 2000), Cdt1 (Maiorano et al., 2000; Nishitani et al., 2000), Dbf4 (Pasero et al., 1999), and MCM proteins (Kneissl et al., 2003). In these events the ORC probably plays the role of a platform or a landing pad (Bell, 2002) on which other factors assemble pre‐RC and also localizes pre‐RC at the origins. Once assembled into pre‐RC, many initiation factors do not need to be bound to the origin ORC to initiate replication. Studies in replicating

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Xenopus extracts revealed that the ORC (and Cdc6) can be removed from chromatin after pre‐RC formation has occurred in G1.The remaining MCM proteins direct the initiation of DNA replication after S phase entry (Hua et al., 1997; Rowles et al., 1999). This finding suggests that the ORC is not required to recruit many of the components that assemble at the origin in an MCM‐dependent manner. Nevertheless the ORC (or its core subunits) ordinarily remains at the origin throughout the G1 and S phases of the cell cycle in Xenopus as well as in budding yeast (DiZey et al., 1994; Rowles et al., 1996) and acts as a chromosomal landmark to identify the site of each origin in subsequent cell cycles. In addition, the constant presence of the ORC at origins suggests that it could be involved in other cell functions such as formation of heterochromatin (see further discussion). It has been shown that the artificial recruitment of any eukaryotic replication initiation factor to a DNA can create a functional origin of replication (Takeda et al., 2005). DiVerent components of the ORC complex and Cdc6 stimulated 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 in the described experiment occurred once per cell cycle and was inhibited by Geminin, indicating that the plasmid was properly licensed during the cell cycle (Takeda et al., 2005). 1. Regulation of ORC Activities To ensure faithful and timely DNA replication cells establish several mechanisms that inhibit the activities of components of pre‐RC following origin activation. The central role of ORC function during the initiation of replication makes it a prime target for cell cycle regulators. The synthesis of the ORC in yeast S. cerevisiae is not highly regulated either at a transcriptional or translational level. However, the mRNA expression of human and Drosophila Orc1 is cell cycle regulated in response to the E2F transcription factor. This regulation confers an additional level of control for the initiation of replication in metazoan cells. In multicellular eukaryotes one or more ORC subunits dissociate from the complex soon after the formation of pre‐RC is complete. This is in contrast to yeast cells where six‐subunit ORCs remain bound to the chromatin during all cell cycle stages. For example, in Drosophila, cellular levels of Orc1 change dramatically during development (Asano and Wharton, 1999). Orc1 in Drosophila embryos accumulates in proliferating cells and only in late G1 and S phases. During mitosis Orc1 is selectively ubiquitinated by APC/Fzr and degraded (Araki et al., 2003; Asano and Wharton, 1999). In mammalian cells, Orc1 is ubiquitinated through the SCF (Skp2) system and in some cases degraded, however, this subsequent degradation is not mandatory (DePamphilis, 2005). It is possible that the diVerences reflect diVerent

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mechanisms of mammalian Orc1 regulation in diVerent cell lines/types. Newly synthesized Orc1 appears as cells exit mitosis (DePamphilis, 2005). Cdk activities have both negative and positive roles in the initiation of DNA replication and cell cycle progression (DiZey, 2004). Numerous studies have shown the role of Cdks in the prevention of rereplication through direct inactivation of pre‐RC proteins. For example, budding yeast ORC is phosphorylated by cyclin‐dependent kinases in a cell cycle‐regulated manner on both the Orc2 and Orc6 subunits (Nguyen et al., 2001; Vas et al., 2001; Wilmes et al., 2004). These modifications to the ORC appear to inhibit new pre‐RC formation after entry into S phase. It is interesting that the simultaneous mutation of the CDK phosphorylation sites on the ORC, elimination of the Cdc6p N‐terminus (preventing its degradation), and constitutive localization of MCM proteins to the nucleus result in reinitiated DNA replication within the same cell cycle, presumably by allowing new pre‐RC formation (Nguyen et al., 2001). Similar studies were performed for S. pombe ORC suggesting that phosphorylation of the ORC negatively regulates reinitiation of replication (Vas et al., 2001). Also, ORC‐dependent association of Cdc2– Cdc13 with replication origins in fission yeast prevents reentry into S phase in the absence of mitosis (Wuarin et al., 2002). Xenopus ORC was found to be phosphorylated in egg extracts in a cell cycle‐dependent way and its chromatin association changes according to its phosphorylation status (Carpenter and Dunphy, 1998; Hua and Newport, 1998; Romanowski et al., 2000; Rowles et al., 1999; Tugal et al., 1998). Drosophila ORC is phosphorylated in vivo and is a substrate for Cdks in vitro (Remus et al., 2005). Both Orc1 and Orc2 subunits are phosphorylated by Cdks in Drosophila. This phosphorylation inhibits ATPase activity of the ORC and its ATP‐dependent DNA‐binding activity. Casein kinase 2 has also been shown to modulate ATP‐dependent ORC–DNA interaction (Remus et al., 2005). The molecular consequences of ORC phosphorylation that inhibits pre‐ RC formation are not completely elucidated. It is likely that modification directly inhibits the association between the ORC and other components of the pre‐RC. In addition, as shown by studies in Drosophila, phosphorylation seems to alter activities of the ORC, such as ATP‐dependent DNA binding and intrinsic ATP hydrolysis by the ORC (Remus et al., 2005). All these activities are required for pre‐RC formation. Overall, this multilevel, cell cycle‐dependent regulation of ORC activity, or ‘‘ORC cycle’’ (DePamphilis, 2005), is a crucial step in preventing rereplication during single cell cycle progression. The ORC does not merely select the sites for assembly of pre‐RC, it is also an important component of multiple cellular pathways that determine when pre‐RCs are assembled. In all eukaryotic organisms ORC subunits undergo cell cycle‐dependent modifications involving phosphorylation and ubiquitination that repress ORC activities during S, G1, and M phases. ORC activity is restored after a completion of mitosis.

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IV. Nonreplicative Functions of the ORC In addition to the DNA replication the ORC is involved in other cell functions. Analysis of ORC binding sites in S. cerevisiae cells revealed that they do not always colocalize with origins of replication. Many of these sites are found within heterochromatic regions, including telomeres. Analysis of ORC localization in Drosophila embryonic and tissue culture cells yielded the same result. The ORC has shown striking preference for heterochromatic regions. It was also found to be important in the number of M phase events, such as chromosome condensation, integrity, and cytokinesis. Another indication that the ORC might be involved in functions other than replication came from the analysis of ORC gene expression in mammalian cells. ORC subunits were expressed in both proliferating and nonproliferating tissues (Quintana et al., 1998; Takahara et al., 1996). All these observations support the involvement of the ORC in functions independent of DNA replication.

A. Gene Silencing and Heterochromatin Formation The first discovered function of the ORC other than the initiation of DNA replication is the establishment of transcriptionally repressed domains at the S. cerevisiae silent mating type loci, HMR and HML (Shore, 2001). In 1984 Miller and Nasmith showed that the establishment of silencing required a passage through the S phase (Miller and Nasmyth, 1984). Moreover, these mating type silencers all contain ACSs and can act as a replication origins on plasmids (Dubey et al., 1991). Therefore, this function of the ORC in silencing did not come as a surprise. A number of genetic screens identified mutations in several DNA replication genes, including ORC genes, which were also defective in silencing (Axelrod and Rine, 1991; Ehrenhofer‐Murray et al., 1995; Foss et al., 1993; Micklem et al., 1993). Other studies indicate that the roles of the ORC in replication and silencing could be separated. Many mutants of ORC genes that distinguished between the function of ORCs in DNA replication and its role in transcriptional silencing have been identified (Bell et al., 1995; Dillin and Rine, 1997; Fox et al., 1995). In more recent studies, silencing was established on episomes that lack origins and are therefore unable to replicate (Kirchmaier and Rine, 2001; Li et al., 2001). In these cases altered silencers were used that replaced the ORC and the ACS with a Gal4pSir1p fusion protein, important for the establishment of silencing, and Gal4 DNA‐binding sites, respectively. These data suggest that neither the generation nor the passage of replication forks is required for the establishment of these silenced loci. However, the requirement for passage through S phase is retained. Other studies found that yeast Orc1 protein has a

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significant similarity to Sir3, the protein factor required for mating‐type silencing. This similarity is especially strong at the N‐terminus and a mutation that removes this domain results in a loss of transcriptional silencing but shows no eVect on DNA replication (Bell et al., 1995). This domain is important in recruiting Sir1, another key factor in the establishment of mating‐type silencing (Fox et al., 1997; Triolo and Sternglanz, 1996). The ORC binds to hundreds of nonsilencer replication origins distributed throughout the yeast genome, but Sir1 binding is confined to silencers (Gardner and Fox, 2001). Studies also suggest that an Sir1–ORC interaction is restricted to silencers, at least in part, through interactions with Sir4, another protein necessary for the nucleation of silencing at the HMR locus (Bose et al., 2004). Studies in Drosophila revealed that the function in the establishment of transcriptionally repressed heterochromatic regions may be a conserved feature of the ORC. Several groups have shown that the ORC has a role in the establishment and/or maintenance of the Drosophila heterochromatin (Huang et al., 1998; Pak et al., 1997). Staining of Drosophila cells and chromosomes with antibody against the Orc2 subunit revealed that Drosophila ORC is enriched at heterochromatic regions of the genome (Fig. 2) where it interacts directly with Hp1, a well‐known modifier of position eVect variegation involved in the formation of heterochromatin (Pak et al., 1997). A similar interaction was also observed between Xenopus ORC and Hp1 proteins. Moreover, mutations in Drosophila ORC result in mislocalization of Hp1 and have a similar Su(var) (supression of position eVect variegation) phenotype (Huang et al., 1998; Pak et al., 1997). In addition to its colocalization with Orc2 in Drosophila cells, Hp1 was found to interact with the N‐terminal domain of the Orc1 subunit in both Drosophila and Xenopus (Pak et al., 1997). Hp1 also colocalizes with the Orc2 subunit in human cells and depletions of Orc2 result in mislocalization of Hp1 (Prasanth et al., 2004). In human cells, however, Orc1 is degraded after entry into S phase (DePamphilis, 2005) at the time when the Orc2, which is associated with Hp1, begins to be restricted to centric heterochromatin (Prasanth et al., 2004). The association of Orc2 with Hp1 in the latter half of the cell cycle when Orc1 is greatly reduced or not present suggests that Orc2 must associate with Hp1 via another interaction. This interaction could be direct, via another ORC subunit or through an ORC/Hp1‐associated protein (Shareef et al., 2001). Hp1 has a bromo‐adjacent homology (BAH) domain and a related ‘‘shadow’’ domain, both of which are important for targeting Hp1 to the chromatin. The same domain is found in multiple Orc1 homologues as well as Sir3 and DNA methyltransferases (Callebaut et al., 1999). Both of these domains of Hp1 are important for the interaction with Drosophila Orc1 (Pak et al., 1997). The chromodomain of Hp1 recognizes histone H3 that is methylated on lysine 9 and is required for Hp1‐induced gene silencing and also for

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FIG. 2 Immunofluorescence of Drosophila Schneider L2 mitotic chromosome spreads (A–C) colocalization of Orc2 and HP1: (A) Orc2 (red); (B) HP1 (green); (C) merge of Orc2 and HP1 (regions of colocalization in yellow). Arrows indicate regions of strong colocalization at the fourth, pericentric X, and Y chromosomes. Arrowheads indicate regions of partial colocalization, such as in the pericentric heterochromatin of chromosome 3, as shown. The asterisk marks the position of intensely autofluorescent debris. (D) DNA stained with Hoechst 33258 (blue), with recognizable chromosomes labeled. (E) Schematic diagram of heterochromatic regions of Drosophila chromosomes as indicated by diVerential dye‐binding fluorescence. Euchromatin is in light gray, moderately staining regions in dark gray, and heavily staining regions in black. The latter two zones also constitute that fraction of a‐heterochromatin rich in satellite DNA (Lohe et al., 1993; Pimpinelli et al., 1985). (F–H) Karyotyped distribution of Orc2: (F) DNA stained with Hoechst 33258 (blue), (G) Orc2 (red), (H) merged DNA and Orc2. Bar ¼ 5 mm. From Pak et al. (1997).

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centromere function in fission yeast and Drosophila (Bannister et al., 2001; Grewal and Moazed, 2003; Kellum and Alberts, 1995). Orc2 depletion in human cells did not aVect this modification (Prasanth et al., 2004) and, therefore, the loss of HP1 from heterochromatin after Orc2 depletion suggests a role for the ORC in recruiting or maintaining HP1 in the heterochromatin. The studies from Drosophila, Xenopus, and human cells on the interaction between the ORC and HP1 (Huang et al., 1998; Pak et al., 1997; Prasanth et al., 2004) suggest that DNA‐binding activities of the ORC might be responsible for recruiting HP1 to heterochromatin, just as the ORC recruits the S. cerevisiae Sir1 heterochromatin protein to specific, transcriptionally silenced loci (Triolo and Sternglanz, 1996).

B. Chromosome Structure, Chromatid Cohesion, and M Phase Events The findings described in the previous section strongly suggest that the ORC is involved not only in chromosome duplication but also in the establishment and maintenance of chromosome structure, suggesting that it may coordinate all stages of the chromosome inheritance cycle. It has been demonstrated that the ORC is involved in positioning of nucleosomes at ARS1 (Lipford and Bell, 2001). In addition, human ORC interacts with the histone acetyltransferase, HBO1 (Burke et al., 2001; Iizuka and Stillman, 1999), suggesting that histone acetylation around origins is an active process in which chromatin is remodeled by replication initiators. The use of the ORC in all these events is probably justified by its regular distribution along the chromosomes (MacAlpine et al., 2004; Wyrick et al., 2001). It is possible that the tight coupling between DNA replication and chromatin assembly favored a selection of proteins associated with origins to recruit chromatin assembly factors required for chromatin formation. DNA replication is tightly coupled to chromatid cohesion and chromatin assembly (Mello and Almouzni, 2001; Skibbens, 2000). The proper segregation of sister chromatids during anaphase depends on the establishment of sister chromatid cohesion during S phase and chromosome condensation before mitosis (Nasmyth, 2002). Biochemical and genetic evidence indicates that establishment of sister chromatid cohesion is also closely linked to DNA replication, most likely mediated by components of the replication fork (Carson and Christman, 2001). Another link between sister chromatid cohesion and DNA replication came with the discovery of an alternative replication fork clamp (RFC) loader complex, mutations of which lead to loss of sister chromatid cohesion (Mayer et al., 2001). Many ORC mutants in budding yeast and Drosophila display defects in passage through M phase. Studies of temperature‐sensitive ORC mutants in

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budding yeast have shown that they could arrest at either G2/M or prior to the initiation of DNA synthesis (Bell et al., 1993; Dillin and Rine, 1998). Interestingly, arrest in G2/M was still observed when the DNA replication/ damage checkpoint proteins Rad9 and Mec1 were mutated (Dillin and Rine, 1998), suggesting that the G2 arrest was not caused by checkpoint pathways involving these proteins. However, if the mutant cells were arrested at M phase and then brought to a nonpermissive temperature and released, cells arrested with a G1 DNA content. Large‐scale synthetic lethal analysis has been used to screen for genetic interactions of the ORC to study its functions in more detail (Suter et al., 2004). It was found that a combination of mutant alleles for Orc2 (orc2‐1) and Orc5 (orc5‐1) with the complete set of haploid deletion mutants revealed synthetic lethal/sick phenotypes with genes involved in DNA replication, chromatin structure, checkpoints, DNA repair, and recombination, and other genes that were unexpected on the basis of previous studies of the ORC. Many of these genetic interactions are shared with the genes that are involved in initiation of DNA replication. Strong synthetic interactions were demonstrated with null mutations in genes that contribute to sister chromatid cohesion. A genetic interaction between orc5‐1 and the cohesin mutant scc1‐73 suggested that the ORC function contributes to sister chromatid cohesion (Suter et al., 2004). Moreover, further experiments linked sister chromatid cohesion genes to silencing at mating‐type loci and telomeres. In Drosophila, flies with homozygous mutations of orc2, orc3, and orc5 genes show obvious defects in DNA replication as well as in chromosome condensation, have elevated levels of chromosome breakage, and have an abnormally high number of cells arrested in metaphase (Loupart et al., 2000; McHugh and Heck, 2003; Pflumm and Botchan, 2001). Studies of Orc2 dynamics in human cells revealed that a portion of Orc2 was localized to centrosomes throughout the entire cell cycle (Prasanth et al., 2004). Depletion of Orc2 protein from human cells produced two distinct cell cycle‐arrested phenotypes. Some cells displayed S phase defects and mislocalization of Hp1. Other cells were arrested in an aberrant mitotic‐like state and contained replicated DNA. These cells also displayed abnormally condensed chromosomes, failed chromosome congression, and multiple centromeres (Prasanth et al., 2004). The question remains as to whether these phenotypes observed in yeast, fly, and human cells are caused by consequences of replication defects and not directly by the ORC. Experiments described previously assessed only the final arrest point of the ORC mutations. Therefore, it is still possible that the failure to complete M phase was due to defects in DNA replication inducing other checkpoint mechanisms, which has nothing to do with a direct role of the ORC in mitosis. For example, the phenotypes of the Drosophila ORC mutations are similar to those of other mutations in DNA replication

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proteins including mutant alleles of Mcm2 and 4, Mcm10, cdc45, and PCNA (Christensen and Tye, 2003; Pflumm and Botchan, 2001). Other studies, however, found that depletions of PCNA, Orc1, and Cdc6 from human cells (Prasanth et al., 2004) and Rfc4 in Drosophila (Krause et al., 2001) did not result in abnormal chromosome condensation. This suggests that specific mitotic phenotypes observed following Orc2 depletion are due to abnormalities in both centrosome and centromere/kinetochore function. Since Orc2 localizes to both structures and disrupts centrosome localization of proteins like CENP‐F, it is possible that ablation of Orc2 has a direct role in causing the phenotype (Prasanth et al., 2004). The association of Orc2 with centrosomes suggests a link between replication of chromosomes and their segregation. Although molecular details are lacking, the initiation of DNA replication appears to be linked to the structure that pulls chromosomes apart and places them at a critical position for future cell division (Prasanth et al., 2004). Thus, in S. cerevisiae, Drosophila, and human cells, a general defect in DNA replication may have consequences for the correct preparation of chromosomes for subsequent mitosis. Although it is unclear how these alterations in DNA replication lead to defects in chromosome condensation, these findings suggest that there are important events that must be coordinated between DNA replication and mitosis.

C. Role of the ORC in Cytokinesis Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. Studies of the Orc6 protein in Drosophila and human cells suggest another connection between the ORC and other events of the cell cycle. The Orc6 gene is the least conserved of the ORC subunits and amino acid alignments with the budding yeast Orc6 and the metazoan smallest subunit show no statistically significant homologies. The Drosophila (Chesnokov et al., 1999) and Homo sapiens (Dhar and Dutta, 2000) Orc6 subunits are homologs and are similar in size to the S. pombe counterpart (Moon et al., 1999), all of which are considerably smaller than the S. cerevisiae Orc6. Moreover, the human Orc6 homolog does not seem to be tightly associated with the other subunits (Dhar et al., 2001a; Vashee et al., 2001), but when expressed in the baculovirus system with the other ORC genes the protein does join a six‐subunit complex (Vashee et al., 2001). Drosophila Orc6 is an essential component of the complex, as it is required for ORC‐dependent DNA binding and replication and an ORC(1–5) complex could not complement an ORC‐depleted extract for DNA replication (Chesnokov et al., 2001).

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Biochemical studies in Drosophila cells indicate that there is a significant excess of Orc6 relative to other ORC subunits (Chesnokov et al., 2001). No other ORC subunits were found in a form unassociated with other ORC proteins. Given the important role that Drosophila Orc6 plays in cell‐ free replication and the other activities of the ORC that were analyzed, it was of interest to determine whether this separate pool of ORC6 is localized with the other ORC subunits in the cell. Figures 3 and 4 show the results of direct immunofluorescence studies of endogenous ORC proteins in Drosophila embryos. Before the onset of cellularization, Orc1, Orc2, and Orc6 subunits were found in a nuclear space during mitosis. During S phase Orc1 was exported from the nucleus and degraded (Fig. 3) (Araki et al., 2003; Asano and Wharton, 1999). Orc2 and Orc6 proteins remained in the nucleus (Fig. 3). However, after cellularization, Orc6 seems to localize in both cytoplasm and nucleus (Fig. 4). The signals for Orc6 could be blocked by preincubating the aYnity‐purified antibodies with recombinant Orc6 proteins and are clearly distinct from the Orc2 pattern (Chesnokov et al., 2001). Studies of endogenous and overexpressed Orc6 protein in Drosophila tissue culture cells also confirmed its preferential localization proximal to the cytoplasmic membranes (Chesnokov et al., 2001). Detailed analysis of Orc6 localization in Drosophila and human cells during diVerent stages of the cell cycle showed the presence of Orc6 at the cell membrane and at the cytokinetic furrow (Chesnokov et al., 2003; Prasanth et al., 2002). During mitosis in human cells Orc6 localizes to kinetochores and to a reticular‐like structure around the cell periphery (Prasanth et al., 2002). During segregation of chromosomes at anaphase, Orc6 is found along the plane of cell division as well as at the midbody before cell separation. Silencing of Orc6 expression by RNA interference resulted in cells with multipolar spindles, aberrant mitosis, formation of multinucleated cells, and decreased DNA replication. Prolonged periods of Orc6 depletion caused a decrease in cell proliferation and increased cell death (Prasanth et al., 2002). These results implicate Orc6 as an essential gene that coordinates chromosome replication and segregation with cytokinesis. In Drosophila, a two‐hybrid screen revealed that Orc6 interacts with Peanut (pnut), a member of the septin family of proteins important for cell division (Chesnokov et al., 2003). Pnut and Orc6 colocalize together in Drosophila embryonic cells (Fig. 5) and in tissue culture cells at membranes and cleavage furrows between dividing cells. This interaction, mediated by a distinct carboxy‐terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with dsRNA resulted in the formation of multinucleated cells and also reduced DNA replication. Deletion of the C‐terminal Orc6–Peanut interaction domain and subsequent overexpression of the Orc6 mutant protein

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resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows (Chesnokov et al., 2003). These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis. The primary question raised by these findings might be posed as follows: Does the role of Orc6 in cytokinesis actually link the regulation of DNA

A

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FIG. 3 In vivo localization of Drosophila ORC subunits in early embryos before cellularization. S phase (A) and mitotic phase (B) embryos are shown. Confocal microscopy was performed using a Carl Zeiss LSM 510 microscope (40 magnification). Immunostaining of the Drosophila embryos was performed using aYnity‐purified antibody raised against Drosophila Orc1, Orc2, and Orc6 proteins. The same Drosophila embryos were used in the upper or lower rows for both (A) and (B).

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dapi B

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FIG. 4 In vivo localization of Drosophila ORC subunits in early embryos after cellularization (A–C). Confocal microscopy was performed using a Carl Zeiss LSM 510 microscope (40 magnification). Immunostaining of the Drosophila embryos was performed using aYnity‐ purified antibody raised against Drosophila Orc2 and Orc6 proteins. Arrows indicate the same cells within the Drosophila embryo shown here. From Chesnokov et al. (2001).

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~4 h of development FIG. 5 Orc6 and Pnut colocalize in Drosophila cells. (A) Immunofluorescence studies of Orc6 and Pnut in Drosophila embryos at the beginning of the cellularization (¼2 h of development). Antibodies against Drosophila Orc6 and Pnut protein were used for staining. 40 ,6‐Diamidino‐2‐ phenylindole (DAPI) staining and merged images are also presented. (a–d) A longitudinal view; (e–h) the corresponding tangential view. (B) Immunofluorescence studies of Orc6 and Pnut in Drosophila embryos at 3.5–4 h of development. DAPI (a), a‐Orc6 (b), and a‐Pnut (c) antibodies were used for staining. A merged image and a magnified section (d and d0 ) are also presented. From Chesnokov et al. (2003).

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replication to this late step in cell division? It looks as if in metazoan cells, Orc6 functions in multiple aspects of the cell division cycle, including DNA replication, chromosome segregation, and cytokinesis (Fig. 6). It may be that Orc6 participates independently in these events. That might explain an excess of Orc6 observed in Drosophila cells. Alternatively, Orc6 could be essential for inducing or signaling multiple cell cycle checkpoints that coordinate the many processes of the cell division cycle. A priori, it could be suggested that after completion of DNA replication Orc6 might dissociate from chromatin and move from the origins of DNA replication into the cytoplasm, to centromeres, and to the cytokinesis apparatus to ensure the correct order and coordination of diVerent cell cycle events. It is also possible that the first steps toward building a prereplication complex in early G1 or at the end of mitosis might be tied to the successful completion of cytokinesis. Orc6 molecules at the cleavage furrow might participate in some event during cytokinesis and then after execution shuttle to chromosomes perhaps with other proteins. This shuttling might make the completion of a cytokinetic function dependent on the start of a new round of replication. During this late step in cytokinesis, Orc6 might couple the cytokinetic and DNA replication pathways. Alternatively, Orc6 may participate in some early role in cytokinesis

FIG. 6 Multiple functions of Orc6 in Drosophila. Orc6 protein is an essential component of ORC in Drosophila. It is required for ORC–DNA binding. It also has a function at the cell membrane where it interacts with Pnut protein. See text for discussion.

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assisting in a targeting function for septins in metazoans, thus potentially linking assembly of such septin rings at the cleavage furrow to the completion of DNA replication. In all these models, chromosome duplication, segregation, and cytokinesis would be coordinated by a common participant.

D. Other Nonreplicative ORC Functions The fly Orc3 protein, a subunit of the ORC complex, had been found to localize to the Drosophila neuromuscular junction (NMJ) and is required for its normal development and function (Pinto et al., 1999; Rohrbough et al., 1999). The latheo (lat) (Orc3 homolog) gene was originally identified from a transposon mutagenesis as a ‘‘memory mutant’’ (Boynton and Tully, 1992). It was shown that olfactory memory was reduced in lat P‐element mutants, although the relevant sensorimotor responses appeared normal. Initial genetic experiments indicated that latP1 is a hypomorphic mutation of an essential gene; null mutations failed to produce any adult flies. Detailed analysis of lat revealed that it is a conserved gene that functions as part of the ORC during DNA replication (Pinto et al., 1999). Mutations in lat cause a failure of cell proliferation during larval development and in the central nervous system of Drosophila, resulting in defects in adult brain structures. Given the molecular, biochemical, and cellular data (Pinto et al., 1999), the most likely explanation for the memory defect of lat mutants would be malfunctions during the development of the adult brain. Interestingly, lat (Orc3) is also expressed in the presynaptic terminals of larval motor neurons (Rohrbough et al., 1999). This observation suggests the more complex possibility that lat may play a separate role in neuronal plasticity in terminally diVerentiated neurons. Other studies (Huang et al., 2005) addressed the expression levels of ORC genes in mouse brain tissues. It was found that ORC genes encoding for subunits 2, 3, 4, and 5 are expressed at high levels in adult mouse brain tissues including cerebral cortex, hippocampus, and cerebellum, whereas the Orc6 gene is expressed at a moderate level. However, no transcripts for Orc1 were detected. This expression pattern is consistent with the structural relationship between the ORC subunits in that Orc2–5 subunits are known to form the core of the ORC and thus seem likely to be coregulated at the transcriptional level. They are selectively localized to the neuronal somatodendritic compartment and enriched in the membrane fraction. siRNA knockdown of ORC subunits dramatically reduced dendritic branch formation and severely impeded dendritic spine emergence. Overexpression of ORC ATPase motif mutants promotes dendritic branching. Huang et al. (2005) suggest that the ORC core complex might have a novel role in regulating dendrite and dendritic spine development in postmitotic neurons.

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Like cytokinesis, dendrite branching and spine formation are very diVerent from the process of DNA replication. However, in vivo imaging studies have found that synaptic growth at the Drosophila NMJ, at least morphologically, is surprisingly similar to yeast budding, the unique cytokinetic process of budding yeast (Zito et al., 1999). This suggests that there may be some common features underlying neuronal morphogenesis and cytokinesis. Indeed, the Orc3 protein has been found to localize to the Drosophila NMJ and orc3 mutants display impaired NMJ development and function (Pinto et al., 1999; Rohrbough et al., 1999). Orc6 function during cytokinesis has also been found to depend on its interaction with a septin protein (Chesnokov et al., 2003), a component well known for its role in organizing the actin cytoskeleton at yeast budding sites. The findings that the ORC may regulate dendrite and spine development by controlling the organization of the actin cytoskeleton are therefore consistent with these observations.

V. Concluding Remarks Since the discovery of the origin recognition complex in yeast cells much has been learned about the initiation of DNA replication and proteins involved in this process. Parallel studies in yeast and metazoan species revealed significant similarities and dissimilarities in the initiation of DNA replication and an assembly of the prereplicative complex at the origins, which starts with the ORC. Conservation among eukaryotic ORCs is limited, allowing the possibility that ORC functions and regulation may vary among diVerent species. Detailed analysis of ORC functions in the last several years began to clarify the mechanisms of its association with the origins, the role of the ORC in the formation of pre‐RC, and the control of ORC activity by ATP. The role of the ORC in other than DNA replication cell functions such as heterochromatin formation and transcriptional silencing, chromosome condensation, and cytokinesis is also an additional example of connections between diVerent regulators of the cell cycle. Other studies implicated replication initiation proteins in cell cycle events diVerent from replication. MCM proteins are required for transcription in addition to their well‐documented role in DNA replication (Snyder et al., 2005). Pre‐RC formation and replication licensing are required for cohesin loading on chromatin during the G1 phase (Gillespie and Hirano, 2004; Takahashi et al., 2004). It would be very interesting to determine if the processes of chromosome cohesion, chromatin condensation, heterochromatin formation, transcription, or cytokinesis are linked with the initiation of DNA replication through the use of shared protein components.

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Acknowledgments I thank Michael Botchan, Pat Higgins, Kirill Popov, and Carl Schmid for discussions and advice on the manuscript. I apologize to all colleagues whose publications could not be individually cited because of length restrictions. Work in the author’s laboratory is supported by a grant from the NIH (GM69681).

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