Gene name - Origin recognition complex subunit 2
Function - Initiation of DNA replication
Keywords - Potential role in chromatin remodeling (See Polycomb and Trithorax genes) - component of Origin recognition complex
Symbol - Orc2
Classification - ORC2 homolog
Cellular location - nuclear
Before discussing Origin recognition complex 2 (ORC), some background about DNA replication and recent research efforts:
DNA replication in eukaryotes takes place in multiple sites of origins of replication scattered throughout the genome. Building an understanding of the proteins involved and the nature of the DNA sequences serving as origins of replication has been a major effort of molecular biologists since the early 1980s. Increasingly, the rewards garnered from this research are being realized. These include an understanding of the relationship between cell cycle checkpoints and DNA replication and an understanding of the link between DNA replication and transcriptional activation and silencing.
A major breakthrough in understanding of DNA origins of replication came in 1979 with the identification in the budding yeast (S. cerevisiae), of autonomously replicating sequences (ARS). ARSs are sequences that allow the transformation (genetic modification) of yeast by plasmids to take place at high frequency. In such transformed cells plasmids are maintained autonomously, as episomes, suggesting that ARSs may act as replication origins.
Subsequently an essential ARS consensus sequence (ACS) was identified. The ACS is an 11 base pair sequence found within all yeast ARSs. The conserved sequence is absolutely required for ARS function; this requirement suggests that ACS serves a critical function in ARSs. A search began for proteins that bind to the critically important ACS. A protein complex, consisting of six subunits, serves as a eukaryotic DNA-synthesis initiation factor: the Origin Recognition Complex (ORC). ORC binds to ACS in an energy dependent manner. Genetic studies have show that the ORC is required for DNA replication (Rowley, 1994 and references).
The Drosophila homolog of the yeast Orc2 gene was found by accident. In studying DNA sequences in proximity to the inositol polyphosphate-1-phosphatase gene, a sequence was found showing homology to budding yeast S. cerevisiae Orc2 (Gossen, 1995).
Having completed the historical detour, what is the Origin Recognition Complex and how does it function? One function of ORC is initiation of DNA synthesis. Mutations in the ORC genes cause a substantial defect in plasmid maintenance. A second function, involvement in transcriptional silencing, is suggested by the fact that ORC binds specifically to the ACS at each of the four silencers of yeast mating type loci HML and HMR. In addition, the establishment of repression at the mating type loci requires passage through S phase. The most convincing evidence that ORC is involved in silencing is that SIR1, one of the four proteins required for silencing, can bind directly to ORC1, the largest of the ORC subunits, and that targeting of SIR1 to ORC1 at a silencer is sufficient to establish a silenced state (Triolo, 1996).
The Drosophila Orc2 gene, expressed in a yeast Orc2 mutant defective for replication and silencing, complements the silencing defect but not the replication defect. This indicates that the replication and silencing functions of ORC are separable. The persistence of the silent state at HMR mediated by Drosophila ORC2 is much less stable during meiosis than it is during mitosis. This indicates an erasure of the silent phenotype during meiosis, and suggests that mitotic inheritance is mechanistically distinct from meiotic inheritance (Ehrenhofer-Murray, 1995).
The ability of Drosophila ORC2 to complement the silencing defect in yeast mutants provides the first evidence that the structural similarity between Drosophila and yeast proteins reflects a functional homology. In addition this result suggests a silencing role for ORC in Drosophila. Although such a role has yet to be proved, this is a promising avenue for future investigation (Ehrenhofer-Murray, 1995).
There is a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. DmORC is found on chromatin at all cell cycle stages of the embryonic syncytium in a diffuse, granular pattern throughout the DNA but is highly concentrated at foci along the apical surface of the interphase nuclei, consistent with the known orientation of pericentric heterochromatin. No differences in DmORC distribution are apparent in embryos after cellularization. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalizes with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 are found in physical complex. DmORC2, 5 and 6 are also found in this complex. Neither DmORC2 nor 6 show reproducible interactions with HP1. The association of ORC1 with HP1 is shown biochemically to require the chromodomain and shadow domains of HP1. Amino acid residues 161-319 of DmORC1 are likely to carry multiple sites of contact with HP1. The amino terminus of DmORC1 contains a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Heterozygous DmORC2 recessive lethal mutations result in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors (Pak, 1997).
The Drosophila Orc2 is developmentally regulated and is most abundant during the earliest stages of embryogenesis, concomitant with the highest rate of DNA replication. Fractionation of embryo nuclear extracts reveals that ORC2 is found in a tightly associated complex with five additional polypeptides, much like the yeast ORC. These studies will enable direct testing of the initiator-based model of replication in a metazoan (Gossen, 1995).
Research into the mechanism of gene amplification can lead to understanding the protein circuitry involved in initiation and maintenance of DNA synthesis. Mutations in Drosophila E2F and in the DP gene coding for E2F's dimerization partner, affect chorion gene amplification and ORC2 localization in ovarian follicle cells. The following section will consider how E2F regulates ORC localization in follicle cells undergoing gene amplification. Ovarian follicle cells undergo a set of mitotic divisions before switching to an endo cycle (a cycle consisting of only S phase and a gap phase) and becoming polyploid. Genomic replication ceases after four endo cycles, but two genomic regions that contain clusters of chorion genes continue to replicate so that the chorion genes are amplified as much as 80-fold relative to genomic DNA. The chorion genes encode the eggshell proteins. Amplification of the chorion genes is needed to produce sufficient chorion protein for a normal eggshell, and amplification occurs by repeated rounds of initiation of DNA replication and fork movement to produce a gradient of amplified DNA extending ~100 kb. Mutants with reduced amplification have a phenotype of thin eggshells and female sterility. Chorion gene amplification appears to use components that are required normally for initiating DNA replication. Mutations in the Drosophila orc2 gene disrupt amplification. Overexpression and inhibition studies indicate that cyclin E is needed for amplification also (Calvi, 1998). Because the levels of Cyclin E protein oscillate with genomic replication but remain constant in follicle cells undergoing amplification, it has been postulated that the high Cyclin E activity blocks genomic replication and that some mechanism permits the amplicons to escape this block to rereplication (Royzman, 1999 and references).
In follicle cells, the ORC2 protein is localized throughout the follicle cell nuclei when they are undergoing polyploid genomic replication, and its levels appear constant in both S and G phases. In contrast, when genomic replication ceases and specific regions amplify, ORC2 is present solely at the amplifying loci. Mutations in the DNA-binding domains of dE2F or dDP reduce amplification, and in these mutants specific localization of ORC2 to amplification loci is lost. Interestingly, a dE2F mutant predicted to lack the carboxy-terminal transcriptional activation and RB-binding domain does not abolish ORC2 localization and shows premature chorion amplification. The effect of the mutations in the heterodimer subunits suggests that E2F controls not only the onset of S phase but also origin activity within S phase (Royzman, 1999).
Minichromosome maintenance proteins (MCMs) are conserved proteins that are essential for the initiation of DNA synthesis in all eukaryotes (see Drosophila Disc proliferation abnormal). Because the MCMs associate with chromatin in an ORC-dependent manner, the localization of the MCMs were examined in follicle cells. MCM2 is located throughout the nucleus and shows staining similar to that seen with MCM proteins in human and Xenopus replication systems. Similar staining was observed with Drosophila MCMs 4 and 5. During the mitotic divisions and subsequent follicle-cell genomic polyploidization, MCM staining appears bright in some follicle cells and faint in others. This staining pattern has been reported previously for embryonic and larval tissues: the bright MCM signal may correlate with binding of MCM to chromatin prior to replication. In contrast to ORC, MCM protein remains nuclear at stage 10 and discrete subnuclear foci are not observed. MCM staining is faint and diffuse in all the follicle cell nuclei of stage-10 egg chambers and all subsequent stages (Royzman, 1999).
Given that cyclin E is an important target of the E2F transcription factor, an attempt was made to determine whether the effects of the dDP and dE2F mutants on chorion gene amplification result from a change in either the levels or the activity of Cyclin E. No change is observed in the levels of cyclin E transcripts in stage 10A or 10B follicle cells from either wild-type or the dDP and dE2F mutant ovaries. A further test was made for an effect of E2F on Cyclin E in follicle cells by monitoring Cyclin E protein in wild-type and mutant ovaries with a monoclonal antibody against Cyclin E. Surprisingly, in dDP and dE2F mutants the levels of Cyclin E protein are normal in follicle cells at all stages, including stage 10B. In the dDP and dE2F mutants there was no effect on Cyclin E staining in follicle cells at any stage. Therefore, the dDP and dE2F mutant effects on chorion gene amplification appear not to occur via Cyclin E. Two other expected transcriptional targets of E2F, PCNA and RNR2, are not induced in the follicle cells of either wild-type or the dDP and dE2F mutants. This suggests that a G1-S transcriptional program is not driving amplification in the follicle cells (Royzman, 1999).
The fact that the levels and activity of Cyclin E are not affected in dDP or dE2F mutant follicle cells suggests that the role of Cyclin E in amplification is either parallel to or upstream of E2F. In evaluating the mechanism by which dDP and dE2F affect ORC localization and DNA replication it is useful to consider each of the three alleles and the distinct effects separately. There are two aspects of ORC localization: clearing of ORC uniformly present in the follicle cell nuclei and subsequent specific localization of ORC to the amplicons. The dDPa1 mutation has the most severe effect in reducing BrdU incorporation and produces eggs with the thinnest shells. In addition, in some egg chambers continued follicle cell polyploidization occurs in place of amplification. The fact that in all the dDP mutant egg chambers nuclear localization of ORC2 persists, and ORC2 is not detectable specifically at amplifying foci could indicate that amplification requires that ORC be cleared from genomic chromatin and assembled at amplification origins. There are two outcomes from persistence of genomic ORC localization. It either blocks amplification or in a minority of egg chambers permits continued genomic replication. The clearing of ORC from genomic origins may be linked to a global change that permits rereplication and amplification of those loci that retain ORC binding (Royzman, 1999).
The dE2Ffi1 mutants have less severe phenotypic effects. ORC is cleared from genomic origins but is not localized to amplification origins. The outcome of this appears to be that genomic polyploidization appropriately stops, but amplification is reduced. These effects also support the idea that ORC concentration at amplifying foci is needed for rereplication. It is proposed that the dE2Ffi1 defect is less severe than that of dDPa1 because a second dE2F gene exists that is able to compensate partially for the dE2F mutant protein (Royzman, 1999).
The absence of an effect of the dE2Ffi2 mutation on ORC localization is consistent with the fact that in this mutant, genomic replication ceases and amplification occurs. It is striking that amplification occurs earlier and has increased levels in mutant flies with a predicted truncated form of dE2F lacking the RB-binding domain. Thus restriction of the onset and extent of origin amplification may be regulated by E2F complexed with RB. It has been demonstrated that RB, when complexed to E2F, is capable of recruiting histone deacetylase and thereby converting chromatin to a compacted state. This state is correlated with inaccessibility to transcription factors, and it is reasonable to propose that it would also hinder binding of replication factors. Thus in this model, E2F in complex with RB would cause histone deacetylation in the vicinity of replication origins, leading to inhibition of amplification until stage 10B. The inability of dE2Fi2 protein to bind RB would prevent inhibition and result in premature amplification (Royzman, 1999).
The differences between the three mutations in the E2F subunits provides insights into the mechanism by which E2F may influence ORC localization. This effect could be direct or indirect. Both the dDPa1 and dE2Fi1 mutations are predicted to weaken E2F DNA binding. Thus the known E2F activities should be present but at reduced levels. For example, these two mutant proteins should retain transactivation activity and the ability to bind RB, repress transcription, and alter chromatin structure. Despite these activities, ORC foci are not detected, implying that the ability of E2F to bind DNA is crucial for its ability to influence ORC localization. This conclusion is supported by the fact that ORC is localized properly in the dE2Fi2 mutant in which the protein has a normal DNA-binding motif and is predicted to lack the transactivation and RB-binding domains (Royzman, 1999).
The suggestion that the critical activity of E2F in controlling ORC localization is DNA binding makes it possible that E2F has a direct interaction with ORC to localize it to amplification origins. There are candidate E2F-binding sites within the amplification control region for the third chromosome cluster. dE2F could not be detected at discrete nuclear foci when amplification is occurring (I. Royzman and T. Orr-Weaver, unpubl. cited in Royzman, 1999); however, dE2F may be more difficult to visualize in situ than ORC. Another alternative is that E2F influences ORC by one of its transcriptional targets. There may be an E2F transcriptional target whose gene product affects ORC localization. Alternatively, the key target might be another subunit of ORC. In human cells ORC1, but not ORC2, is transcriptionally regulated by E2F. The observation that the truncated form of dE2F (dE2Fi2) is sufficient for ORC2 localization would then suggest that dE2F normally activates the transcription of the critical target gene by recruiting another positive regulator to the promoter or by displacing a negative regulator (Royzman, 1999).
The mutations in dDP and dE2F reveal a previously unrecognized role for E2F in controlling replication origin activity within S phase by affecting ORC localization. These results both define a new cell cycle function for E2F and suggest that it affects replication complex assembly directly or via one of its targets. Defining this mechanism will greatly enhance understanding of the regulation and developmental control of replication initiation (Royzman, 1999).
The fly ORC2 has a 30% homology with yeast ORC2, with less pronounced homology at the N-termini (Gossen, 1995)
date revised: 12 May 99
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