DNA Replication

Maria Zannis-Hadjopoulos, McGill Cancer Center, Montreal, Quebec, Canada

Based in part on the previous version of this Encyclopedia of Life Sciences (ELS) article, DNA Replication by Maria Zannis-Hadjopoulos and Gerald B Price.

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Article Contents
. Introduction . Mechanism of DNA Replication . Mammalian DNA Replication . Structure of Mammalian Origins of DNA Replication

Deoxyribonucleic acid replication is the process leading to the duplication of genetic material in an organism.

. Components Associated with Origin Function . Origin-binding Proteins . Origin Usage

Introduction
Deoxyribonucleic acid (DNA) replication is a fundamental biological process that ensures duplication of the genome, and thus its propagation. The DNA structure has been described as a right-handed double helix (B-DNA helix), which indicated that the DNA molecule was capable of self-replication. This replication is semiconservative and, depending on the system, it can be either unidirectional or bidirectional, whereby the nascent strands emanate from a specic initiation sequence called the origin of DNA replication (ori). The replication origin is the cisacting DNA element that denes the site of initiation of DNA replication and provides regulatory control over the process. See also: DNA Replication Origins; DNA Structure

. Conclusions . Acknowledgements

doi: 10.1002/9780470015902.a0005282.pub2

Mechanism of DNA Replication

Owing to the complexity of higher eukaryotic genomes (e.g. the human haploid genome consists of 2.9 109 bp) and the existence of multiple replicons, most of our current knowledge about the mechanisms of DNA replication stems from the study of model systems with simpler genomes such as prokaryotic (plasmids, bacteriophages and bacteria) and eukaryotic (viruses, yeasts and protists) systems. The study of these systems has revealed some universal principles for the replication process. See also: Gene Structure and Organization; Genome Size All DNA replication systems require basic enzymatic activities that include the following: a helicase, which unwinds the double-stranded DNA template and denes the growing point of the replication fork; a topoisomerase, which relieves the torsional strain that develops ahead of the growing fork; the single-stranded binding proteins (SSBs), which coat and stabilize the unwound, singlestranded template DNA and a DNA polymerase, which polymerizes the nascent strand in the 5 to 3 direction. The antiparallel nature of the two strands of the DNA helix and the bidirectional mechanism of replication require a dierent mode of replication for each of the unwound template strands of 5 to 3 replication polarity. The leading strand, unwound in the 3 to 5 direction, is the template for 5 to 3 elongation of the nascent strand, while the lagging strand, which has an antiparallel direction, requires regular

reinitiation of replication. This reinitiation is carried out by the primase activity that synthesizes ribonucleic acid (RNA) primers (initiator RNA, iRNA), whose free 3-OH end is extended by the DNA polymerase. Lagging-strand synthesis involves the production of Okazaki fragment intermediates, whose length varies, from 20003000 bp in prokaryotes to 200300 bp in eukaryotes. The iRNA is later excised by the action of special factors (RNAase H1 and exonuclease FEN-1, also called maturation factor-1, MF1), these gaps are then lled in with deoxyribonucleotides by a DNA polymerase, and the Okazaki fragments are nally joined together by the action of DNA ligase. A consequence of the fact that DNA polymerases extend the primers only in the 5 to 3 direction is that they are unable to copy the 5 ends of the linear chromosome. In the absence of telomerase, the enzyme that maintains telomere length, this would result in the shortening of telomeres after each replication cycle. See also: DNA Helicases; DNA Polymerases: Eukaryotic; Telomeres: Protection and Maintenance; Topoisomerases

Mammalian DNA Replication

Studies involving DNA bre autoradiography suggested that mammalian DNA replication initiates at multiple distinct sites and proceeds bidirectionally outwards from a centrally placed origin. Measurements estimated the number of initiations in a mammalian nucleus at approximately 104 105, serving replicons whose size varied from 50 to 250 kb. Using more recent methodologies, a study based on in vivo labelling with nucleotide analogues and DNA bre spread techniques, estimated the replicon size to be 46 kb on average, ranging from 10 to 200 kb (Takebayashi et al., 2001). Another more recent study, using a novel hybridization assay (genomic Morse code) on single combed DNA molecules from primary keratinocytes, mapped all the detectable initiation zones in a 1.5 Mb region of human chromosome 14q11.2 and found that initiation zones are located in intergenic regions and that only a fraction of
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these zones are actually used in a single cell cycle (Lebofsky et al., 2006). There are more potential replication origins than those activated during a normal S phase. This origin redundancy most likely provides a safety net in the cases where normal replication is perturbed, ensuring genomic stability (Schwob, 2004). The overall rate of replication is determined by the frequency of initiation and the rate of elongation (replication fork progression). The latter process has been well characterized, owing to the development of cell-free (in vitro) replication systems, which are mostly based on plasmids containing the Escherichia coli chromosomal origin of DNA replication, oriC, or the SV40 (simian virus 40) viral replication origin. In vitro yeast and mammalian replication systems have also been developed. Under normal conditions, each eukaryotic origin initiates replication only once per cell cycle. See also: Synchronous and Asynchronous Replication

Structure of Mammalian Origins of DNA Replication

The bacterial replicon model, proposed by Jacob et al. (1963), species that a cis-acting replicator sequence is the target element for the binding of a trans-acting initiator protein (IP), allowing the initiation of replication. The replicon, which was based on the E. coli model, was dened as a genetic element that replicates as a unit, possessing a unique origin of replication that serves as the target of the positive-acting initiator protein. Although eukaryotes, which have increased genomic complexity, initiate replication at multiple sites, the same principles apply. As in prokaryotes, eukaryotic DNA replication is regulated at the level of initiation. Once initiated, replication proceeds at a more or less constant rate until the entire genome has been

duplicated during the S phase of the cell cycle. See also: Cell Cycle Checkpoint Genes and Cancer Replication origins are dened both functionally and genetically, the functional ori being the DNA site where replication begins, while the genetic ori (replicator) is an inheritable sequence required for initiation of replication, as revealed by cis-acting mutations. In simple genomes, these two sites are coincident. Replication origins share a common structure, consisting of an origin recognition element (ORE), a DNAunwinding element (DUE) and one or more binding sites for specic transcription factors (Figure 1). In the budding yeast, Saccharomyces cerevisiae, replicators have been identied as the DNA sequences that serve as origins in both the chromosomes and in plasmids into which they are cloned. They are called autonomously replicating sequences (ARSs), because they allow these plasmids to replicate in yeast cells. In complex genomes, recently developed technology has permitted mapping of DNA replication origins in single-copy sequences in metazoan chromosomes. These studies have shown that in Metazoa, as in systems with simple genomes, DNA replication also begins at specic sites (origins of bidirectional replication, OBR), suggesting conservation of site-specic initiation of replication in all organisms. Further studies present additional supportive evidence for site-specic initiation in mammalian cells. Thus, the same OBR sites of the hamster dihydrofolate reductase (DHFR) region that are used by mammalian nuclei in vivo are selectively activated by a Xenopus egg extract in vitro (Li et al., 2000). Other studies, however, have suggested the existence of initiation zones of 55 kb or more, where initiation occurs without apparent sequence specicity. Using a single-molecule analysis of replicated DNA (SMARD) for the visualization of replication patterns of EpsteinBarr virus (EBV) episomes, a study has concluded that initiation zones rather than

Replication proteins Initiator protein(s) CBP=14-3-3 isoforms Ku & IPs? Transcription factors Inverted repeat (DNA cruciform) AT DUE ORE Aux Core Aux Transcription factors

Figure 1 The principal elements of eukaryotic deoxyribonucleic acid (DNA) replication origins (ori). The minimal ori or core element consists of: a DNA unwinding element (DUE) that is easily unwound; an origin recognition element (ORE), which is bound by the initiator protein(s) (IPs), including the Ku protein; an AT-rich sequence (AT), where DNA bending occurs and an inverted repeat (IR) sequence that has the potential of extruding into a cruciform conformation and as such is bound by a cruciform-binding protein (CBP), which in human cells was identified as belonging to the 14-3-3 protein family. The position of the AT and DUE elements with respect to ORE and IR elements indicates only their inclusion within a predicted core. The core element is flanked by the auxiliary components (Aux), which are binding sites of transcription factors that are specific to particular replication systems and influence the initiation of DNA replication, through their interaction with origin-binding proteins and other replication proteins. (Adapted from DePamphilis ML (1993) Annual Review of Biochemistry 62: 29 63, and based on data regarding mammalian replication oris, described by Novac et al., 2002, 2001).

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individual origins may represent the functional units that regulate DNA replication at the level of individual loci (Norio and Schildkraut, 2001). Research has also suggested the existence of initiation zones, which contain multiple specic initiation sites, in several loci in Metazoa, including the human b-globin, c-myc, DNA methyltransferase (DNMT1) and ribosomal RNA (rRNA) gene loci and the Chinese hamster DHFR, among others (Kamath and Leak, 2001). One proposed model suggests that replication origins have evolved from the bacterial and/or episomal to the multicellular eukaryotic, helping to establish chromatin domains of gene expression and raising the possibility that replication can cause remodelling of chromatin and participate in epigenetic modications. Furthermore, origin interference has been suggested as a mechanism of modulating origin function in human cells, especially in the context of initiation zones (Lebofsky et al., 2006). See also: Epigenetic Factors and Chromosome Organization

proteins (CBPs), in addition to one or more IPs. See also: Chromatin Structure and Domains; Matrix-associated Regions (MARs) and Scaold Attachment Regions (SARs)

Origin-binding Proteins
Origin-binding proteins (reviewed in Zannis-Hadjopoulos et al., 2004) bind specically to their cognate DNA-binding site, the ORE. They initiate replication, either by unwinding the DNA (helicase activity) or through their interaction with other replication proteins. In contrast to prokaryotes, where much is known about the proteins involved in the replication of prokaryotic DNA, relatively little is known about the eukaryotic system. Most of the information about chromosomal DNA replication has been obtained by studying the model replication systems of adenoviruses and papovaviruses, in vivo and in vitro. Considerable information has also been obtained by studying the budding yeast, S. cerevisiae, regarding the controlled activation of replication origins, in relation to the cell cycle. Yeast ARSs contain a 15- to 35-bp ORE that includes a conserved 11-bp AT-rich element (ARS consensus sequence or ACS), as the binding site for its origin recognition proteins. An origin recognition complex, ORC, composed of six polypeptides (ranging from 50 to 120 kDa) has been isolated from yeast cells, which is essential for initiation of DNA replication, acting as a landing pad for the assembly of the pre-replication complex (pre-RC). The genes encoding all six ORC subunits have been cloned and ORC homologues have been identied in invertebrates (Caenorhabditis elegans), plants (Arabidopsis thaliana), ssion yeast, y (Drosophila melanogaster) and humans. In S. cerevisiae, ORC is bound to the replication origins (or ARSs) throughout the cell cycle. The pre-RC assembles during G1 phase of the cell cycle, in preparation for initiation of DNA replication at the origin. In budding yeast, this complex consists of ORC proteins, Cdc6p and the minichromosome maintenance (MCM) proteins (licensing factors). Later, activation of cell cycle-regulated protein kinases (CDKs) guides the licensed origin into S phase. The pre-RC gradually dissociates by releasing Cdc6p and MCM proteins, giving rise to the postreplication complex (post-RC), which persists until the next G1 phase, before another round of replication can occur. ORC plays a critical role in the initiation of replication by positioning nucleosomes adjacent to yeast origins of replication, thus inuencing the assembly of pre-RC and supporting the hypothesis that chromosomal context can signicantly inuence origin function. In Schizosaccharomyces pombe, ORC binds to multiple, specic sites within replication origins and this site-specic binding is determined by the Orc4p subunit. EBV replication from the viral replicator oriP also requires ORC and the human replication machinery, since geminin, an inhibitor of Cdt1, was found to inhibit replication from oriP (Dhar et al., 2001). Cdt1, rst identied in the yeast Sc. pombe, is a
3

Components Associated with Origin Function

Activation of origins is dependent not only on the presence of a specic sequence but also on structural determinants. Chromatin structures are commonly divided into euchromatin and heterochromatin. Euchromatin corresponds to genomic regions that possess actively transcribed genes (or potentially active ones), which are decondensed during interphase. The regulatory sequences in these regions are accessible to nucleases and commonly have unmethylated CpG islands and the core histones H3 and H4 are hyperacetylated on their N-terminal lysine residues. Chromatin modication and regulation occur by posttranslational modications of histone tails through acetylation, methylation, phosphorylation and other modications that direct chromatin structure. Alternatively, adenosine triphosphate (ATP)-dependent chromatin remodelling factors alter histoneDNA interactions, so that proteins can interact with nucleosomal DNA. These chromatin modications enable a uid state of the chromatin in which diverse nuclear processes can occur systematically. There are also certain types of sequences that result in (potential) structures (e.g. bent DNA, DUEs, matrix attachment regions (MARs) and inverted repeats (IRs) or palindromic sequences) that are common to most prokaryotic and eukaryotic replication origins. Cruciform structures have been shown in vivo for prokaryotic, mammalian and viral DNA. Cruciforms aect the degree of supercoiling in DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA and directly interact with proteins. IR or palindromic sequences can switch from the linear to the cruciform conformation and serve as recognition signals for specic regulatory proteins of DNA replication and transcription. Thus, initiation of replication may be regulated by cruciform-specic binding

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conserved replication protein that is necessary for the binding of MCM proteins to replication origins. Orc2p associates specically with oriP, most likely through its interaction with the EpsteinBarr nuclear antigen-1 (EBNA1), which has been found to destabilize nucleosomes at oriP. The mammalian ORC activity, however, appears to be regulated during the cell cycle by the selective dissociation and reassociation of Orc1 protein from chromatin-bound ORC (Li and DePamphilis, 2002). The selective instability of Orc1p likely accounts for the absence of functional ORCs during the MG1 transition in mammalian cells (Natale et al., 2000). The human ORC complex (HsORC) is required for initiation of DNA replication and the loading of initiator proteins on to origins, including Cdc6, Cdt1 and the hexameric MCM complex. Although it specically associates with DNA replication origins in vivo, it lacks sequence specicity in vitro, leading to the proposition that another origin-binding protein may recruit HsORC to origins. Such a role was recently demonstrated for the Ku protein, which was found to bind replication origins in a sequence-specic manner before the assembly of the HsORC (Sibani et al., 2005). Recruitment of ORC to replication origins by sequence-specic DNA-binding proteins has also been observed in other mammalian and viral systems. Thus, two closely related DNA-binding proteins, AIF-C1 and AIF-C2, which bind specically with the origin/promoter of the rat aldolase B (AldB) gene, were also shown to associate with Orc1, implying the recruitment of ORC to replication origins (Saitoh et al., 2002), while Orc2 binds specically to the oriP region of EBV through its interaction with the viral initiator protein EBNA-1 (Dhar et al., 2001). Two studies in S. cerevisiae, one using high-density oligonucleotide microarrays (Raghuraman et al., 2001) and the other using a chromatin immunoprecipitation (ChIP) assay, to enrich for DNA bound by ORC and MCM proteins, using it as a molecular landmark for the identication of origins, followed by hybridization to DNA microarrays (ChIP on chip; Wyrick et al., 2001), identied a total of 332 and 429 sites, respectively, that were predicted to contain replication origins. A comparison of the results obtained from the two methods (Stillman, 2001) suggested that the discrepancy in the number of oris detected might be due to the possibility that initiation may not always occur in an ORC- and MCM-dependent manner, but may also occur at sites of induced DNA breaks. Although replicators of simple and complex genomes may have equivalent functional domains, dierences may exist with regard to recognition of the consensus sequence by their respective IPs. For example, although the yeast ACS is found in mammalian (monkey and human) originrich sequences (ors), it is not essential for origin function. Mammalian DNA replication is a highly complex process, having several unique features that distinguish it from simpler systems, which suggest the involvement of additional recognition proteins. Evidence for multiprotein complexes playing a role in mammalian DNA replication has been accumulating. Replication-competent
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multiprotein forms of DNA polymerase have been isolated from human and murine cells, capable of supporting the in vitro replication of SV40 or human origins of DNA replication and polyomavirus, respectively. Furthermore, two new mammalian origin-binding proteins, OBA/Ku and CBP/14-3-3, have been described.

OBA/Ku
A mammalian origin-binding protein, OBA, has been puried through its ability to interact specically with ors8, a mammalian (monkey) origin of replication, and was identied as being identical to the 86-kDa (or 80-kDa) subunit of Ku antigen (reviewed in Zannis-Hadjopoulos et al., 2004). Ku is a heterodimeric (70/80-kDa) nuclear protein with known functions in DNA repair and V(D)J recombination. It is present in all eukaryotes, suggesting a conserved function. There is increasing evidence for the involvement of Ku in DNA replication, through its ability to bind to replication origins (Novac et al., 2001). It is identical to the DNA-dependent adenosine triphosphatase (ATPase) puried from HeLa cells as part of a multiprotein complex that is needed to support the in vitro SV40 DNA replication. It has been shown to bind to a number of replication origins, including the adenovirus type 2 origin, the minimal origin of the monkey ors8, that comprises the A3/4 sequence, a version of a mammalian consensus sequence (Price et al., 2003), the dihydrofolate reductase (DHFR) Chinese hamster replication origin, orib, the human DNMT1 (DNA-methyltransferase) origin (Novac et al., 2001) as well as the human lamin B2, b-globin and c-myc origins (Sibani et al., 2005). OBA/Ku binds specically to A3/4 (Schild-Poulter et al., 2003), which is also capable of supporting autonomous replication in vivo and in vitro (Price et al., 2003). Quantitative chromatin immunoprecipitation (qChIP) assays, whereby proteins are cross-linked to the DNA in vivo and then precipitated with anti-Ku antibodies, indicate that Ku binds in vivo to replication origins in a cell cycle-dependent manner (Novac et al., 2001) and promotes/stabilizes ORC assembly on to origins (Sibani et al., 2005). Ku also has helicase activity and associates with replication proteins. A recent study that used an optimized tandem anity purication (TAP)tagging method identied several new binding partners of Ku, including the MCM protein family that plays a central role in DNA replication and chromosome maintenance (Bu rkstu mmer et al., 2006). A Ku-like protein from S. cerevisiae, OBF2, binds to the yeast ARS121 origin of replication and supports the formation of a stable multiprotein complex at essential replication sequences. Ku may be involved in DNA replication through its association with DNA-dependent protein kinase (DNA-PK), which phosphorylates several DNA-binding proteins, including replication proteins such as replication protein A (RPA), topoisomerase I and II, SV40 large T antigen, Oct-1 and Ku antigen itself. Although Ku knockout mice are viable, they have a growth defect and Ku802/2 mouse embryonic broblasts (MEFs) have reduced growth potential, prolonged

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doubling time, early senescence, and are more sensitive to DNA damage (Nussenzweig et al., 1996). These phenotypes suggested a new and important function for Ku80 in growth regulation. The viability with a defective growth phenotype of the Ku2/2 mice also suggests the existence of alternative mechanisms, substituting for Ku function, albeit sub-optimally. The small size of the Ku-decient mice, the failure of the cells to proliferate normally in culture, their prolonged doubling time and their premature senescence also suggest a role for Ku in DNA replication (Novac et al., 2001). Unlike mice, the targeted disruption of the human Ku80 locus in HCT116 colon cancer cells resulted in nonviable Ku802/2 cells, while Ku80+/2 haploinsucient cells exhibit a prolonged doubling time (Li et al., 2002) and a prolonged G1 phase (Sibani et al., 2005). These ndings indicated that the replication mechanisms operating in mouse and human dier. The Ku86 locus is essential in human somatic cells, unlike their murine counterparts. The viability of the Ku862/2 murine cells suggests the existence of a redundant, alternative pathway in mice, which is absent or not as eective in humans. See also: DNA Recombination; DNA Repair

CBP/14-3-3
A cruciform-specic binding protein, CBP, has been isolated from human (HeLa) cells and shown to bind to cruciformcontaining DNA at the base of the four-way junction. CBP belongs to the 14-3-3 protein family, a highly conserved family of proteins from plants and invertebrates to eukaryotes, consisting of the 14-3-3 isoforms e, b, g, z and s. DNA cruciform structures have been implicated in regulating the initiation of DNA replication. Using ChIP assay and quantitative real-time PCR analysis, CBP/14-3-3 was found to associate in vivo with the monkey replication origins ors8 and ors12. The association was in a cell cycledependent manner and maximal at the G1/S boundary (Novac et al., 2002). Anti-14-3-3 e, -b, -g, -z and -s antibodies inhibited the in vitro p186 replication, a plasmid containing the minimal replication origin of ors8, by approximately 5080%. These antibodies also interfered with the CBP binding to cruciform DNA. It was concluded that CBP/14-3-3 is a replication origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules and dissociating after origin ring. Recent data, coupled with the apparent function of 14-3-3 proteins in the G1/S transition and the role of cruciform DNA in the initiation of replication, suggest that 143-3 may function in the initiation of DNA replication by regulating the formation or maintenance of the pre-RC through interactions with cruciform DNA (Yahyaoui et al., 2007).

example, specicity of origin selection varies during embryogenesis or tissue development and during a uorodeoxyuridine block or infection by polyomavirus or SV40, as revealed by the decrease in replicon size, suggesting an increase in the number of active replication origins. Clusters of origins and their associated replicons (replication units) are activated at dierent times throughout S phase in a dened spatial and temporal order. Recent comparisons of origin activity among origins of DNA replication between normal human and tumour/ transformed cells revealed an approximate 2- to 3-fold dierence in the activation of replication origins, including origins containing the mammalian consensus sequence (Di Paola et al., 2006) as well as the c-myc origin and NOA3, an origin associated with the 3 region of the z subunit of 14-3-3. Dierential usage of origins (active and potential) may be important for the establishment and/or maintenance of dierent cell types, tissue-specic types and the processes of maturation or malignant transformation states. Also, a 400-bp replication enhancer within the ura4 origin region of Sc. pombe denes the relative activities of three replication origins that are located in this region. This suggests that enhancers may inuence the relative activities of individual origins found in the origin clusters of animal cells, thereby inuencing origin eciency and usage in dierent cell states.

Conclusions
The availability of the human genome sequence and the development of DNA microarrays will impact greatly on the study of DNA replication and will enable the detection of cell cycle-regulated genes, whose products are directly involved in this process. Understanding the dynamics of origin selection, and their activation in normal conditions is a necessity as these data will enable insight into the activation and dysfunction which result in disease. Such ndings will further benet not only our current knowledge of DNA replication but will permit for the emergence of evolving technologies of replication, proteomics and genomics.

Acknowledgements
The author wishes to acknowledge the support of the Canadian Institutes of Health Research and the Canadian Cancer Society. See also: DNA Coiling and Unwinding; DNA Helicases; DNA Polymerases: Eukaryotic; DNA Structure

Origin Usage
There exist more potential initiation sites in the genome than are activated during one normal S phase. For

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Further Reading
DePamphilis ML (1999) Replication origins in metazoan chromosomes: fact or ction? BioEssays 21: 516. Kelly TJ and Brown GW (2000) Regulation of chromosome replication. Annual Review of Biochemistry 69: 829880. Malkas LH (1998) DNA replication machinery of the mammalian cell. Journal of Cellular Biochemistry 30: 1829. chali M (2001) DNA replication origins: from sequence Me specicity to epigenetics. Nature Reviews. Genetics 2: 640645. Pearson CE, Zorbas H, Price GB and Zannis-Hadjopoulos M (1996) Inverted repeats, stem-loops, and cruciforms: signicance for initiation of DNA replication. Journal of Cellular Biochemistry 63: 122. Quintana DG and Dutta A (1999) The metazoan origin recognition complex. Frontiers in Bioscience 4: 805815. Stillman B (1996) Comparison of DNA replication in cells from Prokarya and Eukarya. In: DePamphilis ML (ed.) DNA Replication in Eukaryotic Cells. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Tuteja R and Tuteja N (2000) Ku autoantigen: a multifunctional DNA-binding protein. Critical Reviews in Biochemistry and Molecular Biology 35: 133. Zannis-Hadjopoulos M and Price GB (1998) Regulatory parameters of DNA replication. Critical Reviews in Eukaryotic Gene Expression 8: 81106. Zannis-Hadjopoulos M and Price GB (1999) Eukaryotic DNA replication. Journal of Cellular Biochemistry Supplements 32: 114.

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