DNA Replication: Eukaryotic Origins and the Origin Recognition Complex

DNA Replication: Eukaryotic Origins and the Origin Recognition Complex I Chesnokov and A Svitin, University of Alabama, Birmingham, AL, USA ã 2013 Elsevier Inc. All rights reserved. This article is a revision of the previous edition article by Melvin L. DePamphilis and Cong-jun Li, volume 1, pp. 753–760, ã 2004, Elsevier Inc.

Glossary Autonomously replicating sequence (ARS) A DNA sequence that imparts origin activity to extrachromosomal elements in the presence of appropriate replication proteins, and whose activity is sensitive to genetic alterations. Cdc Cell division cycle protein. A large number of Cdc genes have been identified in eukaryotes that affect various stages in cell division. Cdc6 is the name used in the budding yeast, Saccharomyces. cerevisiae; Cdc18 is the name used in the fission yeast, Schizosaccharomyces pombe. The nomenclature for budding yeast proteins is generally applied to other organisms. Cdk Cyclin-dependent protein kinase. This phosphorylates specific amino acids in proteins, but only when associated with a cyclin protein. Cdt1 A protein encoded by Cdc10-dependent transcript 1 in S. pombe. Cdt1 is the same as RLF-B in Xenopus laevis.

Eukaryotic cells replicate their DNA with remarkable precision during the course of growth and division. Each genomic region should be replicated once and only once per cell cycle to avoid genomic instability. To assure such an accuracy of genome duplication, eukaryotes have evolved a mechanism for the initiation of DNA replication that involves multiple origins of replication (ori) along the chromosomal DNA. DNA replication in eukaryotes is a highly conserved process that begins with the binding of the six-subunit origin recognition complex (ORC) to the origins of DNA replication. ORC binds to origin sites in an ATP-dependent manner and helps in recruiting and in the assembly of additional initiation factors (Cdc6, Cdt1 and mini-chromosome maintenance proteins (MCM) 2–7 helicase complex) to form pre-replicative complex (preRC), thus licensing origin for further replication activity. Before the S phase, the combined action of cyclin-dependent kinases (CDK) and Dbf4-dependent kinases (DDK) results in the loading of additional replication proteins to form the pre-initiation complex (pre-IC). DNA synthesis is initiated after the recruitment of DNA polymerase complexes (polymerase a-primase and polymerase e).

Structure and Diversity of Eukaryotic Origins Replicon Model The basis of our current views on replication initiation stems from replicon model, proposed by Francois Jacob and Sydney Brenner in 1963. In this model, a replicon, defined as a DNA molecule capable of autonomous replication, is characterized by two functional components – initiator and replicator. A trans-acting

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DNA replication origin The DNA site where replication begins; also called an origin of bidirectional replication. Mcm Minichromosome maintenance proteins. These were identified as genes required to maintain plasmids in S. cerevisiae. At least seven of these proteins are involved in DNA replication. Origin recognition complex (ORC) Six different proteins that bind to DNA replication origins and thereby initiate assembly of a pre-replication complex. Pre-replication complex (pre-RC) Fourteen different proteins consisting of ORC, Cdc6, Cdt1, and Mcm(2–7) that form a complex with chromatin during G1 phase of the cell cycle and that become the site where DNA replication begins during S phase. Replicator A DNA sequence that imparts origin activity when translocated to other chromosomal regions, and whose activity is sensitive to genetic alterations.

initiator protein binds to a specific sequence, DNA cis-element called replicator. Upon binding, initiator is transformed into replication-competent form which is followed by a replication initiation at the replicator site. DNA replication starts with the separation of DNA strands (DNA unwinding) resulting in a formation of replication forks moving in opposite directions. Due to the antiparallel nature of the DNA duplex, DNA synthesis occurs continuously on one strand (leading strand) and discontinuously on the other (lagging strand). As DNA polymerases can travel along their templates only in one direction (50 –30 ), the DNA synthesis on a lagging strand results in short DNA fragments (Okazaki fragments) which are joined together by DNA ligase.

Replication Origins in Prokaryotes, Viruses, and Lower Eukaryotes Replicon model was first solidly proven in prokaryotic organisms. In Escherichia coli, initiator protein DnaA binds specifically to the 250-bp AT-rich replicator sequence oriC, resulting in a local ATP-dependent DNA unwinding and replicative DNA helicase (DnaB) loading. This is followed by the assembly of replication fork components, including DNA polymerases, and a replication initiation. Viral origins contain short species-specific sequences. These sequences are bound by a single virus-encoded protein (such as SV40 T antigen). The binding is highly sequence-specific; even mild alterations of the viral origin strongly affect replication. Unlike most eukaryotic origins, viral origins can initiate replication several times during the same cell cycle. Viral origins are functionally independent from neighboring sequences.

Molecular Biology | DNA Replication: Eukaryotic Origins and the Origin Recognition Complex

This represents another difference from eukaryotic initiation sites in which origin activity may be strongly influenced by the context of a surrounding DNA. The most well-studied and characterized eukaryotic replication origins are found in the genomes of yeast cells. In budding yeast, Saccharomyces cerevisiae sites of replication initiation were originally identified in a screen for genomic regions supporting replication of extrachromosomal plasmids which carry them. These regions were termed autonomously replicating sequences (ARS) and were shown to have a significant sequence similarity with each other. A typical ARS has size of approximately 100 bp and consists of 11-bp ARS-consensus sequence (ACS or A element) and another less-conserved 10- to 15-bp element (B element) essential for origin function. Mutations in these two elements affect binding of ORC to the origin. At the same time, many studies suggest that additional factors and epigenetic parameters, such as nucleosome positioning, are also important in defining of origin selection and function. Unlike budding yeast, the origins in other eukaryotic species are significantly less strictly defined. Even in fission yeast, Schizosaccharomyces pombe origins are much larger (500– 1000 bp) and are not conservative in sequence, except for being AT-rich. The latter feature is apparently important for ORC binding, as S. pombe Orc4 subunit contains multiple AT-rich sequence-binding motifs (AT-hooks) at the N-terminal part of the protein. Reduction in AT-content of origins results in delayed initiation, thus illustrating connection between sequence composition and functional characteristics of origin in S. pombe.

Metazoan Origins Difference from budding yeast origin structure is even more obvious in metazoan species where origins are large and do not exhibit any sequence conservation; no sequence elements analogous to yeast ARS were found. Situation is even more complicated by a large size and complexity of the genomes in higher eukaryotes, and consequently, by a significant number of replicons and replication initiation sites (Figure 1). For example,

Prokaryotes

Eukaryotes

Multiple potential origins

Single origin

Less frequent initiation events More frequent initiation events when forks move fast when forks move slow

Single initiation event

Genome is replicated in multiple replicons

Figure 1 Replication origins in prokaryotes and eukaryotes. Bacterial chromosome contains one origin which initiates single replication fork. Multiple potential origins are found on eukaryotic chromosomes. Activation of these origins is coregulated with replication fork progression rate; if forks slow down, additional origins are activated.

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an estimated 30 000 replication origins may function at different times in human cells. Distribution and localization of origins change during development in metazoans, which further obscures the study and an understanding of origindefining factors in these species. Such flexibility of DNA replication in metazoans is well exemplified by the crucial change in this process during the development of insects and amphibians. In Drosophila melanogaster early embryos and Xenopus laevis fertilized eggs, DNA replication is extremely rapid, taking only 10–30 min for complete replication of the whole genome due to large number of functioning origins spaced as close as 4–7 kb. In these conditions, frequent initiation of replication employs practically all available origins. Later in development, origin distribution significantly changes, with the initiation occurring selectively on a fraction of all origins, and time needed for genome replication also increases up to 8–10 h. Some of the most characterized origins are found in D. melanogaster. These origins control an amplification of two clusters of chorion genes in somatic follicle cells that surround developing oocyte. At a specific stage of oogenesis, these two genomic regions on the third and X chromosome are amplified up to 60-fold by supporting extra rounds of DNA replication. This process is followed by high expression of the chorion genes, required for the successful formation of the eggshell. Two cis-regulatory elements important for the amplification were identified within the chorion gene cluster: the 400 bp amplification control element 3 (ACE3) and 1 kb orib. It was shown that Drosophila ORC localizes to the chorion amplification gene cluster in follicle cells, and that ACE3 and orib are sufficient to drive this localization. ORC also binds these sequence elements in vitro, however, in DNA-binding assays, Drosophila ORC shows at best sixfold preference for the chorion locus origin DNA compared to random DNA fragments. This suggests that origin determination in metazoan relies on the mechanisms other that simple initiator–replicator sequence recognition. Many metazoan origins were mapped by different methods in mammalian cells. They can be roughly subdivided into two groups. Origins of one group localize to relatively confined locations on chromosomes. One example of this type of origins is human b-globin locus. This locus includes five genes that encode b subunit of hemoglobin. DNA replication initiation occurs within several kb region located in the intergenic space between d- and b-globin genes. Removal of this initiation region abolishes origin activity. Under these conditions, b-globin locus is passively replicated from outside origin. Initiation region itself contains two independent nonoverlapping replicators, and each of them is sufficient to induce replication in ectopic sites upon transfer. Combination of asymmetric purine–pyrimidine sequence and several AT-rich stretches is required for replication initiation activity of these replicators. The second type of higher eukaryotes origins is characterized by large extended initiation zones, tens of kilobases in size. Initiation in these origins occurs infrequently at multiple sites distributed across the entire region. One of the better studied origins of this kind is Chinese hamster ovary dihydrofolate reductase (DHFR) locus, a 240-kb chromosomal domain amplified in methotrexate-resistant CHO cell line. In this locus, replication initiation events are mapped across the whole 55-kb intergenic region. The initiation of DNA replication is sensitive

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Molecular Biology | DNA Replication: Eukaryotic Origins and the Origin Recognition Complex

to mutations in specific sequences surrounding the initiation zone. For example, the deletion of the DHFR promoter allows initiation to occur within the DHFR gene, which does not normally initiate replication. Removal of specific sequences at the 3’ end of the DHFR gene, causing DHFR transcripts to extend to the intergenic region, confines initiation to the far 3’ end of the locus. Importantly, no consensus origin sequence was found among the metazoan initiation sites. In vitro DNA-binding assays demonstrate that metazoan ORC preferentially binds to sequences with higher AT-content, though it is unable to differentiate between naturally occurring origin sequences derived from known initiation sites and artificial AT-rich DNA fragments. Thus, origin specification in higher eukaryotes apparently involves other factors rather than simple recognition of replicator sequence by initiator.

Specification and Selection of Eukaryotic Origins Mechanisms or Origin Specification Several mechanisms have been described which participate in targeting ORC to the specific sites on a DNA. First, ORC itself has a DNA-binding ability. The binding of ORC to DNA occurs with high specificity in budding yeast, however, in metazoan species, ORC does not have a strong preference for binding to a particular DNA sequence but still favors AT-rich DNA. Second, other replication initiation proteins, such as Cdc6, could enhance ORC interaction with origin sequences. Third, other factors, such as transcription factors or chromosomal proteins, which interact specifically with DNA, could be involved in recruiting ORC to specific sites in the genome. Fourth, characteristics and state of chromatin structure in specific regions of the genome could restrict the areas where ORC can function. Finally, certain specific conditions such as nucleotide pool level or DNA methylation may also influence ORC binding and assembly of the pre-RC. DNA topology may be one of the factors determining a choice of the binding site by ORC. In in vitro DNA-binding experiments, Drosophila ORC demonstrates significant preference for negatively supercoiled DNAs, binding to them with 30-fold higher affinity as compared to linear or relaxed DNA fragments. Such topological sensitivity of ORC binding is closely related to another important factor – a chromatin structure of the binding site. There are several examples of changes in origin usage as a result of alterations in local chromatin structure. In yeast, mutations in histone deacetylase (HDAC) Sir2 facilitate initiation events, rescuing replication mutants involved in pre-RC assembly. In Xenopus and humans, HBO1, a histone acetyltransferase (HAT) directly interacts with the components of pre-RC and is required for MCM loading onto chromatin. Chromatin acetylation was also shown to be critical for chorion gene cluster amplification in Drosophila follicle cells. Influence of chromatin structure on the replication initiation is not restricted to histone acetylation events. In Xenopus system, DNA methylation state was shown to affect initiation. Origin activity is repressed by highly methylated state which interferes with the ORC binding. However, if a low-methylated region is created on such DNA, ORC binding is detected, and replication initiation occurs. Also, DNA methylation was

shown to determine replication start location, either directly or indirectly, in mammalian cells. Transcription is known to induce a negative supercoiling which facilitates DNA unwinding required for the replication initiation. This idea is supported by the data that, replication initiation frequency in fission yeast genome is twice as high in the intergenic regions of divergent transcription, compared to the ones of convergent transcription. Also, formation of active transcription complex can specify replication origin on the plasmid DNA in Xenopus extracts. Preferential localization of replication origins to the actively transcribed regions, also suggests that an open chromatin state associated with these regions is favorable for the replication initiation. Many yeast origins are known to be located in the proximity of promoters. Moreover, direct interactions between ORC and transcription factors E2F1, Rb and Myb complex important for chorion gene expression, are described in Drosophila. Mutations in these genes affect localization of ORC and a function of chorion-cluster amplification origin. Another example of tight transcription–replication association comes from studies in human cells, where Epstein-Barr virus (EBV) transcription factor EBNA1 (EBV-coded nuclear antigen 1) participates in recruitment of ORC to the viral replication origin, oriP. These results could partially explain why all attempts to identify consensus sequence of higher eukaryotes origin have failed; various transcription factors could impart both developmental and DNA sequence specificity to each particular origin. Replication factors such as Cdc6 can also participate in the origin specification. In budding yeast, interaction between ORC and Cdc6 is facilitated by origin DNA and reduces dissociation rate of ORC from origin. In metazoan species, Cdc6–ORC interaction might also enhance DNA binding by ORC. It is possible, that Cdc6 and transcription factors act in combination to specify the interactions between ORC and DNA.

Origin Plasticity Important characteristic of the origin selection process is its dynamical regulation. This feature is illustrated by the origin plasticity phenomenon, described by J. Herbert Taylor in 1977. Under the conditions of thymidine starvation of Chinese hamster ovary cells, when rate of replication fork progression is decreased, total replication rate is compensated by the increase in the number and frequency of replication initiation events; cells with slower fork movement activate additional origins. On the other hand, acceleration of fork progression results in a decrease in the number of active origins. Thus, replication fork movement rate and distances between active origins are coordinately regulated (Figure 1). The number of genomic sites with assembled pre-RC is higher than the number of the sites of actual replication initiation. Pre-RC sites are formed in excess; possibly, to supply a pool of additional origins which can be utilized when needed. This mechanism protects genome from being under-replicated, since the regions which would be out of reach for stalled or slowed replication forks started from usually active origins, would still be replicated due to the activity of the local dormant origins. The mechanism of dormant origins activation could be mainly stochastic. In this model, dormant origins are repressed by passing replication forks which originated from preferred,

Molecular Biology | DNA Replication: Eukaryotic Origins and the Origin Recognition Complex

normal origins. However, under conditions of the replication stress, dormant origins become unrepressed since the replication forks do not reach them. Another model stipulates that stalled replication fork induces conformational changes in the surrounding chromatin and facilitates replication initiation from less preferred dormant origins. In any case, dormant origin activation would be triggered by the changes in replication fork progression.

Methods to Study DNA Replication Current understanding of eukaryotic origin structure, selection, and DNA replication in general was achieved by the employment of a broad array of in vitro and in vivo methods. The most important of them include chromatin immunoprecipitation followed by analysis on microarrays (ChIP-Chip) or direct high-throughput sequencing (ChIP-seq) where ORC or other components of pre-RC are cross-linked to the DNA, isolated by immunoprecipitation and associated DNA is identified. DNA footprinting as well as assays based on identification of early replication intermediates, such as nascent DNA strand assay and 2D gel analysis, are also important in vivo approaches. Important quantitative parameters of replication, such as origin densitites in the genome and rates of replication fork progression, are assessed using DNA fiber-based approaches where chromosomes are pulse-labeled with modified nucleotide precursors and newly synthesized DNA is visualized by immunofluorescence. Cell-free systems that allow studies of DNA replication in vitro are described for humans, Xenopus and Drosophila and can be used for functional dissection of the processes and factors involved in the initiation of DNA replication.

Origin Recognition Complex as the Core of Replication Initiation Machinery ORC Discovery, Conservation, and Structure The identification of the ORC in S. cerevisiae was an important advance in understanding eukaryotic DNA replication. It was identified by Stephen Bell and Bruce Stillman in 1992 as a factor that specifically bound to the yeast ARSs. 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 recruitment of other essential replication factors to the Ori. 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. 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 differences in the structure of eukaryotic origins of DNA replication and an absence of obvious conserved sequences among them. ORC proteins also share homology with another component of pre-RC–Cdc6. This protein is well conserved among eukaryotes and may be a paralog of Orc1. Moreover, Orc1 can be more related to Cdc6 than to other ORC subunits. The structural

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data indicate that ORC and Cdc6 may form a ring-like structure around the DNA reminiscent of MCM helicase ring. Subunits 1–5 of ORC as well as Cdc6 contain conserved Walker A and B ATP-binding domains within the AAAþ fold. These features are characteristic of the proteins which form ring-shaped complexes and bind DNA in the central channel of the ring. The N-terminus of Orc1 contains bromo-adjacent homology (BAH) domain which is important for protein– protein interaction and provides a structural basis for ORC functions in heterochromatin. Orc6, on the other hand, does not share any of the structural features of Orc1-5 and has its own characteristic domains; unique conserved Orc6 protein fold domain at the N-terminus and the coil-coiled motif at the C-terminal part found in metazoan species. This coil-coiled region is important for cytokinetic functions of Orc6.

ORC Functions in Pre-RC Formation Binding of ORC to DNA specifies sites where initiation of replication will occur, and this process is ATP-dependent. Experimental data indicate that ATP binding by ORC, but not the ATP hydrolysis, is important for DNA binding by the ORC. The coordination of DNA and ATP binding by ORC probably involves allosteric interactions among the subunits. It seems that ORC can exist in alternative conformational states induced by stable interaction of ORC with ATP and origin DNA. In humans, it was shown that ATP is also required for maintenance of ORC integrity and serves as a structural cofactor for ORC. Even though ORC is bound at specific chromosomal regions containing origins of replication in both differentiated Drosophila and human somatic cells in vivo, little is known about how ORC complex finds these sequences. Several mechanisms which target ORC to the specific sites on the DNA were proposed. Among those are DNA topology and modifications, chromatin structure, transcription, transcription and replication factors, and nucleotide pool levels. These mechanisms of ORC targeting to specific DNA sites are addressed in more detail above (see section Specification and Selection of Eukaryotic Origins). Although ORC plays a central role in the initiation of DNA replication in all eukaryotes, the interaction of this protein complex with replication origins in other species differs in some respects from that observed in S. cerevisiae. Fission yeast S. pombe has larger and more complex origins than S. cerevisiae. These origins contain multiple AT-rich elements important for its function. The S. pombe Orc4 subunit contains an N-terminal domain that binds to origin DNA even in the absence of other ORC subunits. This domain contains nine copies of the AT hook motif found in a number of DNA-binding proteins. The AT hook motif is known to bind to the minor groove of AT tracts. 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. ATP-dependent ORC binding to DNA provides basis for the sequential recruitment of a number of additional replication factors which together form pre-RC (Figure 2). The recruitment of Cdc6 and Cdt1 occurs first, followed by the repeated

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Molecular Biology | DNA Replication: Eukaryotic Origins and the Origin Recognition Complex

2 Pi ORC

MCM2-7

Cdc6

ATP

Cdt1

ADP

Figure 2 Assembly of the pre-RC. Binding of ORC to the replication origins marks the beginning of pre-RC assembly. On the next step, Cdc6, and then Cdt1, join origin-bound ORC. These proteins together facilitate initial association of MCM2-7 helicase with chromatin. Hydrolysis of ORC- and Cdc6-bound ATP results in loading of MCM2-7 onto DNA.

mammalian Orc1 regulation in different cell lines/types. Cdk activities have both negative and positive roles in the initiation of DNA replication and cell-cycle progression. Numerous studies have shown the role of Cdks in the prevention of rereplication through direct inactivation of pre-RC proteins. The molecular consequences of ORC phosphorylation that inhibit pre-RC formation are not completely elucidated. It is likely, that modification directly inhibits association between ORC and other components of the pre-RC. Overall, this multilevel, cell-cycle-dependent regulation of ORC activity is a crucial step in preventing re-replication during single cell-cycle progression. ORC does not merely select the sites for assembly of pre-RC; it is also an important component of multiple cellular pathways that determines when pre-RCs are assembled. In all eukaryotic organisms, ORC subunits undergo cellcycle-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.

Nonreplicative Functions of ORC loading Mcm2-7, a hexameric-helicase complex responsible for unwinding parental DNA strands. ORC, together with Cdc6 and Cdt1, emerges as a part of a helicase loading machinery that facilitates the assembly of multiple Mcm2-7 complexes which play a crucial role in origin function. Specifically, a coordinated ATPase activity of ORC and Cdc6 facilitates tighter loading of initial MCM2-7 helicase onto the origin DNA along with recruitment and binding of additional MCM2-7 complexes. MCM2-7 loading concludes formation of the pre-RC as shown in Figure 2. At this point, origin is licenced for further activity and ready to proceed to pre-IC assembly. As cells continue through the G1 phase toward S phase transition, additional replication factors are recruited to the origin including CDKs and DDKs, Cdc45, Mcm10, GINS complex, the three eukaryotic DNA polymerases, and the eukaryotic ssDNA-binding protein, RPA. In the absence of ORC, the assembly of the preRC fails in both cells and cell-free systems, even so ORC by itself has only been shown to interact directly with a small subset of these factors. In these events, ORC not only serves as a landing pad but also actively participates in the assembly of pre-RC at the origins. Interestingly, the artificial recruitment of eukaryotic replication initiation factors, such as ORC and/or Cdc6, to a DNA can create a functional origin of replication.

Regulation of ORC Activity Regulation of replication initiation and pre-RC formation is an important mechanism of preventing re-replication and avoiding genomic instability. As a critical component of these processes, ORC becomes the target of several regulatory pathways. In multicellular eukaryotes, one or more ORC subunits dissociate from the complex soon after the formation of pre-RC is complete. For example, in Drosophila, Orc1 subunit is selectively ubiquitinated by APC/Fzr and degraded. In mammalian cells, Orc1 is ubiquitinated through the SCF(Skp2) system and in some cases degraded, however, this subsequent degradation is not mandatory, suggesting several mechanisms of

Many studies indicate that the functions of ORC extend beyond DNA replication. The first discovered nonreplication function for ORC was its participation in the establishment of transcriptionally silent chromatin domains at the budding yeast silent mating type loci (HMR and HML) through the interaction with the silent chromatin protein Sir1. ORC’s function in the establishment of transcriptionally repressed regions appears to be a conserved feature, as both Drosophila and mammalian ORC interact with HP1, a well-known modifier of position effect variegation. Mutations in the Drosophila Orc2 gene suppress position effect variegation and alter the localization of Hp1. siRNA knockdown of Orc2 in human cells results in delocalization of HP1. Human ORC also interacts with the histone acetyl transferase, HBO1 suggesting that histone acetylation around origins is an active process in which chromatin is remodeled by replication initiators. Another nonreplication function of ORC is related to mitotic events in the chromosomes. Different ORC subunits in yeast, Drosophila, and human cells are involved in events such as sister chromatid cohesion, chromosome condensation and chromatid segregation in the mitosis, and mutations in ORC subunits cause defects in these processes. In both Drosophila and human cells, the smallest subunit of ORC, Orc6, has been implicated in coordinating cytokinesis with pre-RC formation and chromosome segregation, a role that it performs independently of the rest of the complex. Orc6 had been found at the cell membranes and cytokinetic furrow in Drosophila and mammalian cells. RNAi/siRNA knockdown of Orc6 in either Drosophila or human cells results in reduction of DNA synthesis, and also in the prominent appearance of cells that have completed mitosis without cytokinesis (multinucleate cells), a phenotype that is not seen following knockdown of other ORC subunits. In Drosophila, Orc6 interacts and colocalizes with the Pnut protein, a member of the septin complex which is required for cytokinesis and other processes that involve spatial organization of the cell cortex. Experimental data suggest that Orc6 may be involved in the regulation of septin complex activities such as guanosine triphosphate hydrolysis and filament formation, and thus control dynamic

Molecular Biology | DNA Replication: Eukaryotic Origins and the Origin Recognition Complex

cytoskeleton events driven by septins. Also, involvement of ORC component into cytokinesis may be important for coordination between replication and subsequent mitotic events.

Concluding Remarks Recent years of research brought significant progress in our understanding of replication–initiation events. Though basic principles of this process are universal, species-specific differences in certain components and steps of replication–initiation can be significant. This is further complicated by the plastic and adjustable character of replication. Such flexibility exists on multiple levels, from connection between replication fork progression rate and density of active origin in the genome, to diverse global patterns of replication in various tissues and on several developmental stages. Many significant questions related to the understanding of replication–initiation mechanisms and their modifications in different species are still open. Among the most important of them are the exact criteria of origin specification, selection, and regulation. What determines whether a particular locus would be an origin and whether this origin would be active or dormant? How exactly is the activation of dormant origins achieved? What determines the timing of origin firing? The role of ORC in other than DNA replication cell functions, such as heterochromatin formation and transcriptional silencing, chromosome condensation and cohesion, and cytokinesis is an additional example of connections between different regulators of the cell cycle. In-depth study of ORC and pre-RC components functions which span beyond replication, can potentially clarify important global regulatory mechanisms connecting different seemingly isolated pathways, such as replication and chromosomal M phase events, cytokinesis, and transcriptional silencing.

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See also: Molecular Biology: DNA Polymerase b, Eukaryotic; DNA Replication Fork, Eukaryotic.

Further Reading Arias EE and Walter JC (2007) Strength in numbers: Preventing rereplication via multiple mechanisms in eukaryotic cells. Genes and Development 21(5): 497–518. Bell SP (2002) The origin recognition complex: From simple origins to complex functions. Genes and Development 16(6): 659–672. Bowers JL, Randell JC, Chen S, and Bell SP (2004) ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication. Molecular Cell 16(6): 967–978. Chesnokov IN (2007) Multiple functions of the origin recognition complex. International Review of Cytology 256: 69–109. Clarey MG, Botchan M, and Nogales E (2008) Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping. Journal of Structural Biology 164(3): 241–249. DePamphilis ML, Blow JJ, Ghosh S, et al. (2006) Regulating the licensing of DNA replication origins in metazoa. Current Opinion in Cell Biology 18(3): 231–239. Duncker BP, Chesnokov IN, and McConkey BJ (2009) The origin recognition complex protein family. Genome Biology 10(3): 214. Gilbert DM (2004) In search of the holy replicator. Nature reviews Molecular Cell Biology 5(10): 848–855. Herrick J and Bensimon A (2008) Global regulation of genome duplication in eukaryotes: An overview from the epifluorescence microscope. Chromosoma 117(3): 243–260. Machida YJ, Hamlin JL, and Dutta A (2005) Right place, right time, and only once: Replication initiation in metazoans. Cell 123(1): 13–24. Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, and Oda M (2010) Eukaryotic chromosome DNA replication: Where, when, and how? Annual Review of Biochemistry 79: 89–130. Randell JC, Bowers JL, Rodrı´guez HK, and Bell SP (2006) Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2–7 helicase. Molecular Cell 21(1): 29–39. Remus D and Diffley JF (2009) Eukaryotic DNA replication control: Lock and load, then fire. Current Opinion in Cell Biololgy 21(6): 771–777. Schepers A and Papior P (2010) Why are we where we are? Understanding replication origins and initiation sites in eukaryotes using ChIP-approaches. Chromosome Research 18(1): 63–77. Tuduri S, Tourrie`re H, and Pasero P (2010) Defining replication origin efficiency using DNA fiber assays. Chromosome Research 18(1): 91–102.