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The Origin Recognition Complex: from Simple Origins to Complex Functions Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW The origin recognition complex: from simple origins to complex functions Stephen P. Bell1 Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA Eukaryotic DNA replication is an intricate process that sites represented true chromosomal replicators and over- requires the coordinate and tightly regulated action of lapped with their associated origins of replication (for numerous molecular machines. Eukaryotic cells must review, see Bell 1995). Mutational analyses of ARS ele- choose hundreds if not thousands of origins of replica- ments identified an essential, conserved, 11-bp element tion and assemble multiple proteins or protein com- associated with all ARS elements known as the ARS plexes at these sites. During S phase, these complexes consensus sequence (ACS or “A element”), as well as are activated in a reproducible, temporal program to gen- multiple 10–15-bp “B-elements” that also contribute to erate twice this number of replication fork machines origin function. The conserved nature of the ACS made dedicated to DNA synthesis. Moreover, these machines it a likely target for a eukaryotic initiator and many ef- must overcome the highly packaged nature of the eu- forts were made to identify such a factor. ORC was ul- karyotic genome to access all DNA sites, without excep- timately identified based on its affinity for the ACS and tion. was therefore a strong candidate for the eukaryotic ini- The key factor in the selection of initiation sites and tiator (Bell and Stillman 1992). DNA binding by this six- the assembly of the replication machinery is the initiator protein complex (referred to as Orc1p–Orc6p, from larg- protein. Originally proposed by Brenner, Cuzin, and Ja- est to smallest subunit) requires ATP (see below), and it cob based on genetic findings, the replicon model hy- is likely that this requirement as well as the low-abun- pothesized the existence of two essential components dance of ORC thwarted previous attempts to identify required for replication initiation: the initiator and the this factor. replicator (Jacob et al. 1963). The initiator is a positively Abundant evidence now supports the conclusion that acting factor that recognizes a specific DNA sequence ORC is the S. cerevisiae initiator. Point mutations in the within the genetic element required for initiation, called ACS that eliminate ORC binding in vitro and in vivo the replicator. The replicator usually overlaps the site of also result in loss of replicator activity supporting the replication initiation, the origin of replication. Subse- hypothesis that ORC mediates the function of this criti- quent studies in Escherichia coli, other bacteria, and cal element (Bell and Stillman 1992; Li and Herskowitz many bacteriophages and eukaryotic viruses identified 1993; Aparicio et al. 1997; Tanaka et al. 1997). Mutations initiator proteins and confirmed this basic model. Un- in the genes encoding the subunits of the ORC complex like these organisms, each of which uses a single repli- result in DNA replication defects and prevent the asso- cator to duplicate their genome, eukaryotic chromo- ciation of all other replication factors at the origin (Bell somes each require multiple replicators to ensure com- et al. 1993; Foss et al. 1993; Micklem et al. 1993; Santo- plete replication of their genome in a timely manner. canale and Diffley 1996; Dillin and Rine 1997, 1998). Although it was possible that multiple initiators func- Finally, in vivo footprinting, protein–DNA cross-linking, tioned in this more complex situation, studies over the and chromatin precipitation studies all indicate that last 10 years have demonstrated that a single conserved ORC is bound to S. cerevisiae origins of replication eukaryotic initiator directs all of these initiation events: throughout most if not all of the cell cycle (Diffley et al. the origin recognition complex (ORC). 1994; Aparicio et al. 1997; Liang and Stillman 1997; The well-defined replicators of Saccharomyces cerevi- Tanaka et al. 1997). siae provided the critical tools to identify ORC. These Unlike the simple replicators of S. cerevisiae, replica- sequences were originally identified by their ability to tors in other eukaryotic organisms remain poorly defined confer stable propagation to yeast episomes and were (for review, see Bielinsky and Gerbi 2001; Gilbert 2001). called autonomous replicating sequences (ARSs). Subse- The most obvious difference between replicators in S. quent studies provided definitive evidence that these cerevisiae and other organisms is at the level of size. Even in the single cell eukaryote, Schizosaccharomyces pombe, replicators are found to be 5- to 10-fold larger 1E-MAIL [email protected]; FAX (617) 253-4043. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ than their S. cerevisiae counterparts. Replicators derived gad.969602. from multicellular eukaryotes are still less well defined GENES & DEVELOPMENT 16:659–672 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org 659 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Bell and only a few have been mapped in any detail. In mul- exact targets and consequences of these modifications ticellular organisms the number and location of origins remain unclear. In addition to activating existing pre- are regulated during development. Particularly dramatic RCs, the elevation of CDK activity also plays a key role examples of such changes in origin usage are observed in preventing new pre-RC formation until the following during development of Drosophila melanogaster and S phase (for review, see Bell and Dutta 2002). In this Xenopus laevis. The rapid DNA replication observed in review, I will discuss first what we know about the role the early embryos of these species is mediated by tens of of ORC in each of these events. Then, I will describe thousands of origins spaced by as little as 4–7 kb (Blu- studies addressing other roles for ORC in the cell. When menthal et al. 1974). Later in development, inter-origin relevant, ORC derived from different organisms will be distances increase, reducing the number of replicators distinguished using the initials of the genus and species used by Ն10-fold (Hyrien et al. 1995; Sasaki et al. 1999). names preceding ORC (e.g., SpORC is S. pombe ORC). Interestingly, in vitro experiments using Xenopus egg extracts indicate that there is little or no sequence speci- ficity required for the initiation of DNA replication ORC and origin specification (Coverley and Laskey 1994). Indeed, although numerous Studies of ORC across a wide range of eukaryotic species studies have pursued the identification of essential ele- indicate that localization of this complex is the initial ments within other replicators, as of yet, no element step in determining the sites of DNA replication initia- with similar properties to the S. cerevisiae ACS has been tion. Despite this, the mechanism by which ORC is lo- identified in any other eukaryote. calized to origins of replication remains incompletely Despite differences in their structure and a lack of ob- understood. In S. cerevisiae, the ACS and B1 elements vious conserved sequences among eukaryotic replica- play a large part in directing ORC to origins of replica- tors, ORC is a conserved feature of replication initiation tion, however, these sequences are unlikely to be suffi- in all eukaryotes tested. ORC-related genes have been cient to direct ORC localization independently. In other identified in organisms ranging from S. pombe to plants eukaryotes, sequences that direct ORC binding are un- to humans (for review, see Dutta and Bell 1997). Muta- known. Indeed, in some cases there appears to be little or tional analysis of ORC-related genes in S. pombe and D. no DNA sequence selectivity for the location of origins. melanogaster both reveal defects in DNA replication (Muzi-Falconi and Kelly 1995; Grallert and Nurse 1996; Landis et al. 1997; Pinto et al. 1999; Loupart et al. 2000; DNA binding by ScORC Pflumm and Botchan 2001). Similarly, immunodepletion studies using X. laevis replication competent extracts The most well characterized activity of ORC is its abil- indicate an absolute requirement for ORC to initiate ity to bind origin DNA. Although initial studies identi- DNA replication (Carpenter et al. 1996; Romanowski et fied the ACS as the S. cerevisiae ORC (ScORC) binding al. 1996; Rowles et al. 1996). Although no genetic evi- site (Bell and Stillman 1992), further studies demon- dence indicates that ORC functions in human chromo- strated that there was at least one additional interaction somal DNA replication, plasmids replicating in human within the adjacent B1 domain (Rao and Stillman 1995; cells by virtue of an Epstein Barr Virus (EBV) replicator Rowley et al. 1995). Detailed studies of the contacts be- are defective in cells carrying an ORC2 mutation (Dhar tween ORC and origin DNA support an extensive inter- et al. 2001b). This, together with other evidence, indi- action between ScORC and the origin and identify addi- cates that EBV uses the human replication initiation ma- tional sequences that interact with ORC between the A chinery, and suggests that HsORC functions in human and B1 region (Lee and Bell 1997). Interestingly, these DNA replication. Thus, even in multicellular organisms
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