Interactive Fly, Drosophila

Transcriptional silencing of the HM mating-type loci in the yeast S. cerevisiae is caused by the localized formation of an altered chromatin structure, analogous to heterochromatin in higher eukaryotes. Silencing depends on cis-acting sequences, termed silencers, as well as several trans-acting factors, including histones H4 (See Drosophila Histone H4) and H3, proteins RAP1 and ABF1, and the four SIR proteins (SIR1-4). Each of the four HM silencers contains an autonomously replicating sequence (ARS) to which the origin replication complex (ORC) binds. This six-protein complex is required for initiation of DNA replication, as well as for silencing. Efficient establishment of the silenced state requires both passage through the S phase of the cell cycle and SIR1 protein. SIR1 can bind directly to ORC1, the largest of the ORC subunits; targeting of SIR1 to ORC1 at a silencer is sufficient to establish a silenced state. SIR1 is known to interact with SIR4, bringing SIR4 in close proximity to SIR3, which is bound by the neighboring RAP1. RAP1 encodes a context dependent regulatory protein that functions as a repressor of transcripion (Triolo, 1996 and references). SIR3 and SIR4 are known to interact with the N-termini of Histone H3 and H4, suggesting a link between the SIR proteins involved in silencing and histones, the major component of chromatin (Hecht, 1995).

The origin recognition complex (ORC), a multisubunit protein identified in S. cerevisiae, binds to chromosomal replicators and is required for the initiation of cellular DNA replication. Complementary DNAs (cDNAs) encoding proteins related to the two largest subunits of ORC were cloned from various eukaryotes. The cDNAs encoding proteins related to S. cerevisiae Orc1p were cloned from the budding yeast Kluyveromyces lactis, the fission yeast S. pombe, and human cells. These proteins show similarity to regulators of the S and M phases of the cell cycle. Genetic analysis of orc1+ from S. pombe reveals that it is essential for cell viability. The cDNAs encoding proteins related to S. cerevisiae Orc2p were cloned from Arabidopsis thaliana, Caenorhabditis elegans, and human cells. The human ORC-related proteins interact in vivo to form a complex. These studies studies suggest that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication (Gavin, 1995).

Transcriptional silencing at the HMRa locus of Saccharomyces cerevisiae requires the function of the origin recognition complex (ORC), the replication initiator of yeast. Expression of a Drosophila melanogaster Orc2 complementary DNA in the yeast orc2-1 strain, which is defective for replication and silencing, complemented the silencing defect but not the replication defect; this result indicates that the replication and silencing functions of ORC are separable. The orc2-1 mutation mapped to the region of greatest homology between the Drosophila and yeast proteins. The silent state mediated by DmOrc2 is epigenetic; it is propagated during mitotic divisions in a relatively stable way, whereas the nonsilent state is metastable. In contrast, the silent state is erased during meiosis (Ehrenhofer-Murray, 1995).

A novel S. cerevisiae Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays (Hardy, 1996).

The origin recognition complex (ORC) is a six protein assembly that binds S. cerevisiae origins of replication and directs DNA replication throughout the genome and transcriptional silencing at the yeast mating-type loci. Cloning of the genes encoding the 120 kDa (ORC1), 62 kDa (ORC3: see Drosophila Latheo), and 56 kDa (ORC4) subunits of ORC is reported in this paper and the reconstitution of the complete complex after expression of all six subunits in insect cells. Orc1p is related to Cdc6p and Cdc18p, which regulate DNA replication and mitosis, and to Sir3p, a regulator of transcriptional silencing. The N-terminal region of Orc1p is highly related to Sir3p, and studies of Orc1p/Sir3p chimeric proteins indicate that this domain is dedicated to the transcriptional silencing function of ORC (Bell, 1996).

In fission yeast, Cdc2 kinase (see Drosophila cdc2) has both positive and negative roles in regulating DNA replication, being first necessary for the transition from G1 to S phase and later required to prevent the re-initiation of DNA replication during G2. Cdc2 interacts with Orp2, a protein similar to the Orc2 replication factor subunit of S. cerevisiae origin recognition complex (ORC). ORC binds chromosomal origins and is essential for chromosomal replication initiation. Fission yeast Orp2 is required for DNA replication and interacts with the rate-limiting replication activator Cdc18. Cells lacking Orp2 undergo aberrant mitosis, indicating that Orp2 is involved in generating a checkpoint signal. These findings suggest that ORC functions are conserved among eukaryotes and provide evidence that Cdc2 controls DNA replication initiation by acting directly at chromosomal origins (Leatherwood, 1996).

The origin recognition complex (ORC), a six-subunit protein, functions as the replication initiator in the yeast Saccharomyces cerevisiae. Initiation depends on the assembly of the prereplication complex in late M phase and activation in S phase. One subunit of ORC, Orc5p, is required at G1/S and in early M phase. Asynchronous cells with a temperature-sensitive orc5-1 allele arrest in early M phase. In contrast, cells that are first synchronized in M phase, shifted to the restrictive temperature, and then released from the block, arrest at the G1/S boundary. The G1/S arrest phenotype can not be suppressed by introducing wild-type Orc5p during G1. Although all orc2 and orc5 mutations are recessive in the conventional sense, this dominant phenotype is shared with other orc5 alleles and an orc2 allele. The dominant inhibition to cell-cycle progression exhibited by the orc mutants was restricted to the nucleus, suggesting that chromosomes with mutant ORC complexes are capable of sending a signal that blocked initiation on chromosomes containing functional origins (Dillin, 1998).

The origin recognition complex (ORC), first identified in Saccharomyces cerevisiae (sc), is a six-subunit protein complex that binds to DNA origins. Here, the identification and cloning of cDNAs encoding the six subunits of the ORC of Schizosaccharomyces pombe (sp) is reported. Sequence analyses reveal that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts from S. cerevisiae, Xenopus, Drosophila, and human. In contrast, both spOrc3 and spOrc6 subunits are poorly conserved. The C-terminal region of spOrc4 is also conserved whereas the N terminus uniquely contains repeats of a sequence that binds strongly to AT-rich DNA regions. Consistent with this, extraction of S. pombe chromatin with 1 M NaCl, or after DNase I treatment, yield the six-subunit ORC, whereas extraction with 0.3 M resultes in a five-subunit ORC lacking spOrc4p. The spORC can be reconstituted in vitro with all six recombinant subunits expressed in the rabbit reticulocyte system. The association of spOrc4p with the other subunits requires the removal of DNA from reaction mixture by DNase I. This suggests that a strong interaction between spOrc4p and DNA can prevent the isolation of the six-subunit ORC. The unique DNA-binding properties of the spORC may contribute to understanding of the sequence-specific recognition required for the initiation of DNA replication in S. pombe. The strong DNA-binding property of the spOrc4p RGRP motif appears to be involved in the recognition of the unique structure of the narrow minor groove of AT-rich regions rather than a specific nucleotide sequence. Fission yeast autonomous replication sequence (ARS) elements have multiple AT-rich regions that are required for ARS function but no specific essential sequence equivalent to the ACS of S. cerevisiae. Therefore, it is possible that the spORC strongly binds to origin sequences through the multiple AT-hook motifs of spOrc4p and that extraction with 0.3 M NaCl is not ample to dissociate the spOrc4p-DNA complex but can release the other subunits as a five-subunit complex (Moon, 1999).

An interaction between the origin recognition complex (ORC) and Cdc6p is the first and a key step in the initiation of chromosomal DNA replication. The assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae is described. Cdc6p increases the DNA binding specificity of ORC by inhibiting non-specific DNA binding of ORC. Cdc6p induces a concomitant change in the conformation of ORC and either mutations in the Cdc6p Walker A and Walker B motifs, or treatment with ATP-gamma-S, inhibit these activities of Cdc6p. These data suggest that Cdc6p modifies ORC function at DNA replication origins. On the basis of these results in yeast, it is proposed that Cdc6p may be an essential determinant of origin specificity in metazoan species (Mizushima, 2000).

Cdc6p is a key factor for the regulation of initiation of DNA replication, but the biochemical function of this protein has been unclear. Studies in vivo suggest that Cdc6p participates in the recruitment of the MCM protein to the pre-RC. It is suggested that it is in this complex that Cdc6p recruits the MCM proteins. Human Cdc6p was shown to contain an intrinsic ATPase activity. Several biochemical activities of Cdc6p that may contribute to its role in initiation of DNA replication have been uncovered. The first of these demonstrates that ORC and Cdc6p interact directly with each other (Mizushima, 2000).

The second type of Cdc6p biochemical activity modulates the DNA binding activity of ORC by restricting DNA binding to functional origin sequences. The increased DNA binding specificity of ORC appears to be accomplished by Cdc6p increasing the rate of dissociation of ORC from nonorigin DNA sequences and by decreasing the rate of association of ORC to nonorigin DNA. Both ATP and a functional nucleotide-binding motif within Cdc6p are required for Cdc6p blocking nonspecific DNA binding of ORC to DNA, suggesting that Cdc6p ATPase activity may be required for this activity. At present it is not known whether Cdc6p is involved in ORC loading on origin DNA in vivo. In vivo footprinting and CHIP assays suggest that ORC is localized to origins throughout the cell division cycle. But it is not known whether both sister chromatids bind ORC at origins immediately after DNA replication, when Cdc6p is not present (Mizushima, 2000).

It is not known when newly synthesized ORC complexes bind to DNA during the cell division cycle in vivo and, therefore, it is not clear when the cooperativity between ORC and Cdc6p results in increased DNA binding specificity. In S. cerevisiae, all six ORC subunits are present at constant levels throughout the entire cell cycle. However, Cdc6p is an unstable protein that varies in amount during the cell cycle, with the protein made just prior to entry into mitosis or in late G1 phase of the cell cycle just before S phase. In rapidly proliferating cells, Cdc6p first binds to chromatin as cells exit mitosis, but in cells that come out of stationary phase Cdc6p is loaded in late G1, prior to activation of the cyclin-dependent protein kinases. Thus it is possible that ORC and Cdc6p interact and bind to vacant origins late in the cell division cycle. The fact that Cdc6p hinders ORC binding to nonorigin sequences in vitro suggests that one function of Cdc6p is to restrict replication only to functional origins. This activity of Cdc6p would increase the probability that all origins have the potential for forming pre-RCs after anaphase of mitosis (Mizushima, 2000).

Cdc6p remains bound to chromatin, most probably via its interaction with ORC, throughout the G1 phase of the cell cycle. During this time, a window exists that allows the MCM complex to bind to origins and form the pre-RC (Mizushima, 2000).

A third biochemical function intrinsic to Cdc6p is its ability to remodel the ORC in vitro so that certain ORC subunits become hypersensitive to protease digestion. The ORC remodeling activity of Cdc6p also seems to require ATP and a functional nucleotide-binding motif in Cdc6p, suggesting that an ATPase of Cdc6p mediates the ORC remodeling (Mizushima, 2000).

It is possible that the conformational change in ORC is involved in recruitment of the MCM proteins and thus, formation of the pre-RC. In support of this suggestion, genetic analyses suggest that ATP binding and possibly ATP hydrolysis activities of Cdc6p are required for the MCM loading on chromatin. Thus it is possible that the remodeled form of ORC is essential for MCM loading, and the role of Cdc6p in MCM loading is indirect via its interaction with ORC. Alternatively, both proteins might cooperate to recruit the MCM complex (Mizushima, 2000).

The identity of the ORC subunits that are remodeled is of considerable interest. On the basis of DNA cross-linking measurements, Orc2p and Orc6p appear to be located on the B2 element proximal side (relative to the A domain) of ARS1, adjacent to the site of initiation of DNA replication. This region of the origin is most likely the site of initial unwinding of the double helix. Although it is not known where the MCM proteins interact with the origin DNA, it is likely that it is near the B2 element for a number of reasons. (1) The MCM proteins have intrinsic DNA helicase activity. (2) The B2 element region of ARS1 has an intrinsic ability to unwind in supercoiled plasmid DNA. Thus it is possible that the MCM protein complex is loaded on to the origin DNA near the B2 element by interacting with ORC that has been remodeled by the ATPase activity of Cdc6p. (3) Because Orc1p, Orc2p, and Orc6p, but not other subunits of ORC are phosphorylated in vitro by the S phase cyclin-CDK Cdc28/Clb5, phosphorylation of these subunits after START may affect the ORC-Cdc6p-ARS DNA interaction and/or the pre-RC (Mizushima, 2000).

Cdc6p, also a target of the cyclin-CDKs, is released from the ORC-origin complex and degraded after cells commit to a new round to DNA replication after passage through START. Therefore, another possibility is that release of Cdc6p from the pre-RC triggers a conformational change in ORC that allows subsequent events during initiation (Mizushima, 2000).

The biochemical activities of Cdc6p in modulating ORC function suggest a way to alter the frequency of origins along chromosomes that has been observed during development in Drosophila and Xenopus. At early stages of development, where origins are close together and the ORC to DNA ratio is relatively high, Cdc6p may not be as important for determining origin specificity. At later stages of development when origins are less frequent along chromosomes, Cdc6p could be an essential component in origin determination. Initial studies with purified ORC from both Drosophila and mammalian ORC suggests that they have relatively weak DNA intrinsic binding specificity. On the basis of the results with yeast ORC, it is suggested that in other eukaryotic species Cdc6p functions with ORC to determine the location of DNA replication origins in chromosomes (Mizushima, 2000).

All processes involving DNA in eukaryotes must contend with the packaging of the genome into nucleosomes and higher order chromatin structures. DNA replication is no exception, and several lines of evidence support a connection between chromatin structure and DNA replication: (1) euchromatic regions of the genome replicate early in S phase, whereas heterochromatic regions replicate late in S phase; (2) dramatic changes in replication origin usage during Drosophila and Xenopus development are tightly correlated with the onset of extensive zygotic transcription and global chromatin restructuring; (3) chromatin remodeling activities (e.g., FACT, CHRAC, and histone acetylases) from several eukaryotes have been implicated in the DNA replication process. Together, these data suggest that chromatin affects the selection and activation of DNA replication origins; however, a molecular understanding of these connections is lacking (Lipford, 2001 and references therein).

Studies of replication origin function in the yeast S. cerevisiae support the hypothesis that chromatin structure exerts a significant influence on DNA replication. Numerous studies indicate that the chromosomal context can significantly alter origin function. For example, many potential origins of replication function well in the context of a plasmid, but poorly or not at all in their normal chromosomal position. Similarly, the time within S phase that each origin initiates replication is strongly influenced by chromosomal context. Studies of the nucleosomal arrangement surrounding origins of replication also suggest a role for chromatin structure during replication initiation. Mapping of nucleosomes surrounding the ARS1 origin of replication indicates that ~180 bp overlapping the core origin sequence elements are free from nucleosomes. In contrast, the adjacent DNA is packaged into positioned nucleosomes. A similar structure is observed at the HMRE origin of replication. Mutations that allow nucleosomes to occupy one or more of the origin elements of ARS1 disrupt origin activity. Notably, moving these nucleosomes back to their normal position through the action of the alpha2 repressor restores normal origin function. These results suggest that nucleosomes interfere with the ability of an origin to initiate replication when they overlap the origin; however, it remains possible that nucleosomes also play a positive role in regulating replication initiation. The mechanisms by which adjacent chromosomal regions influence origin function, the determinants of the precise nucleosomal configuration surrounding yeast origins, and the roles that this configuration may play in replication initiation remain unclear (Lipford, 2001 and references therein).

The ARS1 nucleosome-free region harbors four DNA elements (named A, B1, B2, and B3) that are important for origin function. Two of these elements, A and B3, adjoin the boundaries of the nucleosome-free region. The B3 element is the binding site for the multifunctional DNA binding factor, Abf1p. This factor likely serves as one nucleosomal boundary at ARS1, as mutations in B3 allow nucleosomes to encroach into the origin region. The essential and conserved A element functions with the B1 element as the binding site for the yeast initiator, the origin recognition complex (ORC), and is adjacent to the opposite boundary of positioned nucleosomes at ARS1. The proximity of the ORC binding site to the boundary of the ARS1 nucleosome-free region suggests that ORC influences the position of nucleosomes adjacent to ARS1 (Lipford, 2001 and references therein).

The role of ORC in the positioning of nucleosomes at yeast origins is unclear; however, much has been learned about ORC and its role in replication initiation. ORC is an essential, six-subunit, ATP-regulated complex that is conserved throughout eukaryotes. The association of ORC with DNA is the first step in the assembly of the multifactor, prereplicative complex (pre-RC) at origins of replication prior to initiation. The formation and the components of this complex are not fully understood, but studies in several different eukaryotes have demonstrated that ORC is required to recruit the essential pre-RC components, Cdc6p and the minichromosome maintenance (MCM) proteins, to the origin. Cdc6p is a conserved member of the AAA+ family of ATPases that includes Orc1p and replication clamp loaders, and is required to load MCM proteins onto replication origins. The MCM complex is composed of six essential subunits, each of which contains a conserved ATP binding and hydrolysis motif. The colocalization of MCM proteins with DNA polymerases during S phase, the presence of helicase activity in purified MCM preparations, and the requirement of MCM activity for replication elongation all support the hypothesis that MCM proteins function as the replicative DNA helicase. Nevertheless, the exact role of the MCM proteins at the replication fork is unclear, and may involve functions in addition to helicase activity (Lipford, 2001 and references therein).

To address how replication factor assembly influences and is influenced by adjacent chromatin structures, a molecular analysis of the interaction between nucleosomes and replication factors has been undertaken in the yeast, S. cerevisiae. In vitro chromatin assembly assays and in vivo assays for nucleosome positioning were used to address the determinants of the chromatin structure at origins. It has been demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function (Lipford, 2001).

A model is favored in which the binding of Abf1p or the lac repressor to a second Abf1p binding site (B3x2) or the lac operator (LacO) affects replication by altering the position of one or more positively acting adjacent nucleosomes. In support of this model, a correlation is observed between the extent of the shift in the pattern of Mnase cleavage induced by insertion of the Abf1p and Lac repressor binding sites and the extent of inhibition of replication initiation in these mutations. An alternative model suggests that the binding of Abf1p or lac repressor reduces origin function by sterically hindering pre-RC formation. However, several lines of evidence argue against this possibility: (1) the inserted binding sites are 70 bp away from the ORC binding site, whereas the normal Abf1p binding site (the B3 element) at ARS1 is 66 bp from the B1 element and does not inhibit replication complex assembly; (2) in vivo footprinting analysis of the sequences protected by the pre-RC shows no protection over the region occupied by the second Abf1p binding site or LacO; (3) this and previous studies of the chromatin structure surrounding ARS1 demonstrate that the nonorigin DNA adjacent to ORC is packaged into a nucleosome. Since this positioned nucleosome does not inhibit replication function, it is unlikely that the more distal Abf1p would act in this manner. Instead, it is suggested that this adjacent nucleosome, possibly in collaboration with ORC, acts to recruit one or more components of the pre-RC (Lipford, 2001).

These data (suggesting that alteration of the ORC-dependent nucleosome) configuration of a yeast origin compromises origin function by disrupting pre-RC formation support an expanded, positive role for nucleosomes at origins. Although the possibility of defects in the association of other pre-RC components cannot be eliminated, the finding that assembly of the MCM complex is affected by the nucleosomal alteration is consistent with a role for the adjacent nucleosomes in recruiting this replication factor to ARS1. Such a relationship between the MCM complex and nucleosomes is supported by in vitro studies that detect a tight interaction between mammalian MCM proteins and histone H3. Several lines of evidence suggest that the MCM complex plays a role in replication elongation. Suggestions of interactions between MCM proteins and histones or nucleosomes are consistent with a model that the MCM complex functions in elongation as a chromatin-remodeling complex as well as a helicase. In support of this hypothesis, MCM genes and chromatin-remodeling genes contain related ATPase motifs. Alternatively, the interaction with histones and nucleosomes could represent a function involved only in recruitment or stabilization of MCM protein association with the origin (Lipford, 2001).

In contrast to the facilitation of initiation proposed in this report, previous studies have substantiated a negative influence of nucleosomes on initiation and elongation of replication. Several studies have correlated the presence of a nucleosome over an origin with a loss of origin function. The simplest explanation for these results is that nucleosomal occupancy of an origin obscures cis-acting elements from interaction with initiation factors. Indeed, forcing a nucleosome over the A and B1 elements of ARS1 eliminates initiation, most likely by interfering with ORC binding. In support of this model, it has been found that ORC cannot cooccupy DNA associated with nucleosomes. Interestingly, chromatin-remodeling activities like ChRAC and histone acetylases can facilitate the access of initiation factors to origins when nucleosomes are present. Similar activities (e.g., FACT) may also aid replication elongation through nucleosomes. It will be interesting to determine whether chromatin-remodeling activities facilitate pre-RC formation, replication initiation, or replication elongation in eukaryotic cells (Lipford, 2001).

The proposed dual role of nucleosomes in replication is reminiscent of their dual role in transcription. Analogous to their negative influence on replication, nucleosomes can inhibit transcription by obscuring the binding sites for transcription factors, and by inhibiting elongation by RNA polymerase. Numerous studies have proposed that chromatin-remodeling activities (e.g., Swi2/Snf2 family, histone acetylases, and FACT) alter the structure of nucleosomes to promote transcription factor binding and transcriptional elongation. In addition to their inhibitory effects, nucleosomes can also facilitate transcription. For example, the tandem binding of hormone receptors and transcription factors at the MMTV promoter only occurs when the promoter DNA is wrapped in a nucleosome. In addition, the positioning of a nucleosome in the vitellogenin B1 promoter of Xenopus may stimulate transcription in a chromatin context. A similar scenario may explain the positive role of nucleosomes positioned by ORC in replication initiation. ORC and the adjacent, positioned nucleosomes may create a structure that recruits or stabilizes the MCM complex (or another pre-RC component) at the origin. This type of role for a nucleosome at origins is analogous to the proposed role for a specialized nucleosome in kinetochore formation at S. cerevisiae centromeres (Lipford, 2001).

The dual role of nucleosomes in replication provides a mechanism by which global changes in chromatin structure can influence origin function. For example, during embryonic development in Xenopus and Drosophila, replication origins reside at multiple closely spaced sites. As development proceeds, origins become restricted to a limited set of sites. This transition coincides with the onset of zygotic transcription and extensive chromatin rearrangement. The restriction of origins could be facilitated by the dual role of nucleosomes in origin function. At sites where origins become inactive, nucleosomes may be positioned (e.g., by the transcription apparatus) such that they interfere with the binding of ORC or other replication factors. In addition, ORC-dependent nucleosome positioning may facilitate the formation of newly specified origins. Given that many of these newly specified origins coincide with promoters and intergenic regions, the interplay between ORC-dependent nucleosome positioning and positioning mediated by the transcription machinery may play an important role in defining origins (Lipford, 2001).

ORC-dependent nucleosome positioning may also participate in the role of ORC in the formation of heterochromatin and the influence that chromosomal context has on origin activity in yeast. Recent studies have determined that heterochromatin in yeast is maintained in a unique nucleosomal configuration. Despite the presence of ORC at HMR and HML, the adjacent nucleosomes are positioned in a manner distinct from those at ARS1, and their positioning is dependent upon the silencing protein, Sir3. Therefore, the lack of ORC-dependent nucleosome positioning may inhibit origin function and explain why heterochromatic regions are replicated late in S phase. Similarly, nucleosome positioning that competes with ORC positioning at late-replicating or chromosomally inactive origins may interfere with origin activity at these sites. Detailed analysis of the chromatin structure surrounding such origins as well as mutants that change these structures will be required to test this possibility in the future (Lipford, 2001).

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC). This six-subunit complex, first isolated in Saccharomyces cerevisiae, is evolutionarily conserved. ORC participates in the formation of the prereplicative complex, which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence (ACS) constitutes part of the ORC binding site. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed. The amplification origin (ori) II/9A of the fly, Sciara coprophila has been analyzed. A distinct 80-base pair (bp) ORC binding site has been identified and the replication start site located adjacent to it has been mapped. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, these findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation (Bielinsky, 2001).

CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6

In Saccharomyces cerevisiae cells, B-type cyclin-dependent kinases (CDKs) target two origin recognition complex (ORC) subunits, Orc2 and Orc6, to inhibit helicase loading. Helicase loading by ORC is inhibited by two distinct CDK-dependent mechanisms. Independent of phosphorylation, binding of CDK to an 'RXL' cyclin-binding motif in Orc6 sterically reduces the initial recruitment of the Cdt1/Mcm2-7 complex to ORC. CDK phosphorylation of Orc2 and Orc6 inhibits the same step in helicase loading. This phosphorylation of Orc6 is stimulated by the RXL motif and mediates the bulk of the phosphorylation-dependent inhibition of helicase loading. Direct binding experiments show that CDK phosphorylation specifically blocks one of the two Cdt1-binding sites on Orc6. Consistent with the inactivation of one Cdt1-binding site preventing helicase loading, CDK phosphorylation of ORC causes a twofold reduction of initial Cdt1/Mcm2-7 recruitment but results in nearly complete inhibition of Mcm2-7 loading. Intriguingly, in addition to being a target of both CDK inhibitory mechanisms, the Orc6 RXL/cyclin-binding motif plays a positive role in the initial recruitment of Cdt1/Mcm2-7 to the origin, suggesting that this motif is critical for the switch between active and inhibited ORC function at the G1-to-S-phase transition (Chen, 2011).

Non-transcriptional control of DNA replication by c-Myc: MCM2-MCM7 subunits, ORC2, Cdc6 and Cdt1, were present in the affinity-purified Myc complex

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. This study shows that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions (Dominguez-Sola, 2007).

Minichromosome maintenance (MCM) proteins are part of the pre-replicative complex, a multiprotein complex essential for the assembly and activity of DNA replication origins13. Indeed, all MCM2-MCM7 subunits, ORC2, Cdc6 and Cdt1, were present in the affinity-purified Myc complex, consistent with the known interaction of Myc with MCM2 and MCM7. In contrast, proteins involved in DNA replication elongation (MCM10, RPA and PCNA) were absent. The interaction with pre-replicative complex components was also observed with N-Myc. Other proteins forming complexes with Myc, such as TRRAP18, were not found in this Myc and pre-replicative-complex-associated complex, whereas small, non-stoichiometrical amounts of Max (Myc-associated factor X) were detectable (Dominguez-Sola, 2007).

Myc and pre-replicative complex proteins co-sedimented in high molecular mass fractions (approx1.7 MDa) after glycerol density gradient sedimentation and size-exclusion chromatography of Myc-bound protein complexes. Notably, Myc is also present in a distinct set of fractions that contained the majority of Max protein that co-purified with this complex). These fractions also contained MCM5, which might be involved in other transcriptional complexes. Overall, these results identify a novel Myc-associated complex in mammalian cells that contains pre-replicative complex components and thus suggests a direct role of Myc in DNA replication (Dominguez-Sola, 2007).

It has been proposed that Myc promotes G1/S transition and DNA replication through the transcription of factors promoting S-phase entry and/or cell growth. The current results indicate that Myc control of DNA replication is not dependent on its transcriptional activity in both Xenopus extracts and mammalian cells. Nonetheless, transcriptional regulation of critical target genes may also be an important component of the overall role of Myc in regulating DNA replication initiation. Notably, the transactivation domain of Myc is required to control both DNA replication initiation and transcriptional activity, suggesting that Myc may use a common molecular mechanism to facilitate both DNA transactions. This mechanism might involve Myc-dependent chromatin modifications such as histone acetylation, which might also be implicated in the selection of replication origins (Dominguez-Sola, 2007).

The results indicate that Myc deregulation generates DNA damage and may promote genomic instability by inducing DNA replication stress, strengthening previous observations. This notion is also supported by the dependence on Werner RecQ helicase for Myc-driven proliferation (Grandori, 2003), and by the requirement for RecQ helicases during replication stress. These observations can explain the occurrence of genomic alterations, such as gene amplification and illegitimate replication of some loci, that are consistently associated with Myc deregulation during tumorigenesis. However, in contrast with other oncogenes that may cause DNA re-replication when deregulated, overexpression of Myc increases the number of active replication origins in the absence of detectable re-replication (Dominguez-Sola, 2007).

The results also suggest that the p53-dependent G2/M checkpoint and subsequent apoptosis observed in mammalian cells carrying deregulated Myc alleles may be due to DNA damage generated predominantly during S phase. Frequent p53 inactivation in tumours carrying deregulated Myc genes may then reflect selection for tumoural cells with disabled checkpoint responses. Thus, these results suggest that Myc may exert its oncogenic function, at least in part, by promoting origin activity, thereby inducing replication stress and genomic instability (Dominguez-Sola, 2007).

Xenopus Origin recognition complex

Continued: see Origin recognition complex subunit 2: Evolutionary homologs part 2/2

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