disc proliferation abnormal


Transcriptional Regulation

The function of the neuronal differentiation gene daughterless is required for the proper initiation of neuronal lineage development in all peripheral nervous system (PNS) lineages following the selection of neuronal precursor cells. Previous studies have shown that the ubiquitously expressed Da protein is required for the proper expression of neuronal precursor genes and lineage identity genes in the PNS of Drosophila embryos. These genes are required for differentiation and cell fate determination in the developing PNS. These findings, however, do not explain the failure of the nascent PNS precursors to undergo a normal cell cycle and divide in da mutants. Four genes whose products are required for various stages of the cell cycle are misexpressed in the PNS of da mutant embryos. Cyclin A, barren, disc proliferation abnormal and Histone H1 transcripts are significantly reduced or undetectable in the precursors of the PNS at stages 11 and 12. Precursors are still present at these stages in da mutants. This suggests that all aspects of PNS precursor differentiation examined so far are under the transcriptional control of da. Sensory organ precursors lacking Da may fail to express and/or accumulate other factors, such as critical differentiation genes, required for SOP entry into the cell cycle. It should be pointed out that these factors are unlikely to be the thus-far described neuronal precursor genes, as mutations in these genes do not result in any obvious cell cycle defects (Hassan, 1997).

Protein Interactions

There are at least two distinct types of 600-kDa complexes that contain Drosophila MCM complexes: one contains DmCDC46 (MCM5), homologous to yeast CDC46, and one appears to contain both DmMCM2 and Disc proliferation abnormal (a CDC54/MCM4 homolog). These complexes are stable throughout embryonic division cycles, are resistant to treatments with salt and detergent, and are present during development in tissues undergoing mitotic DNA replication as well as endoreplication. When extracts are prepared under low salt conditions all three MCM proteins co-immunoprecipitate. DmDC46 is found in another complex of about 200 kDa that is especially prevalent in adult flies. Anti-DMCDC46 antibody detects a complex of about 600kDa in Xenopus egg extracts, indistinguishable on native gels from the 600-kDa Drosophila complex. Human homologs of CDC46, CDC54 (homologous to DPA) and MCM2 have been reported to exist in two distinct complexes: one containing CDC46 and the other containing CDC54 to which MCM2 is loosely associated. Thus, partnership in complex formation appears to be evolutionarily conserved (Su, 1996).

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. Three Drosophila MCM proteins have been characterized: DmMCM2, DmMCM4, and DmMCM5. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Cyclin:cdks can prevent an assembly of proteins called the "prereplicative complex" on origins of DNA replication. The prereplicative complexes are thought to contain MCMs. Their absence from chromatin is thought to contribute to the inability of the post S phase nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, a test was carried out as to whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B shows that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. Thus MCM-chromosome association is delayed when mitosis is followed by a prolonged G-1 phase. dacapo mutant embryos lack an inhibitor for cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. It is proposed that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. Thus, it is suggested that cyclins have both positive and negative roles in controlling MCM-chromatin association (Su, 1997).

Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase

The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, Cdc45 protein was purified from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps, and it was found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese) tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. BLAST searches revealed four members of the GINS complex in Drosophlia are CG14549, CG9187, CG18013, and CG2222 (Sld5, Psf1, Psf2, and Psf3, respectively). RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication (Moyer, 2006; full text of article).

This study provides evidence that Cdc45 exists in a stable biochemical unit with the Mcm2-7 and GINS complexes and that this large complex has associated with it an ATP-dependent helicase activity. The evidence that the Mcm2-7 complex is responsible for this activity (as opposed to another helicase or a subset of the Mcm proteins) is not definitive, but it is an attractive hypothesis. Reconstitution of the complex from recombinant proteins will be the next step in testing this notion. The identification of the Cdc45/Mcm2-7/GINS (CMG) complex and its associated helicase activity supports reports that implicate the Mcms, Cdc45, and GINS in chromosome unwinding, and it begins to provide a molecular model for the mechanism of DNA unwinding at the eukaryotic DNA replication fork (Moyer, 2006).

Cdc45 and GINS first associate with replication origins at the G1 to S phase transition after the activation of the cyclin-dependent kinase (Cdk) and Cdc7 protein kinases, and it is possible that phosphorylation of one or more of the CMG complex members by Cdk and/or Cdc7 may promote Cdc45 and GINS association with Mcm2-7. Mcm proteins have been shown to be phosphorylated at the G1 to S phase transition, and Cdc45 and GINS have been shown to preferentially associate with Mcms during S phase. Furthermore, the bob-1 mutant, an allele of Mcm5 in S. cerevisiae, suppresses the requirement for the Cdc7 kinase, and it is tempting to speculate that this mutation bypasses a modification that is essential for CMG complex assembly (Moyer, 2006).

The exclusive assembly of Cdc45 and GINS with Mcm2-7 at a prereplication complex just before and during DNA synthesis would also clarify why only a small percentage of the total Cdc45, GINS, and Mcm2-7 proteins in the starting nuclear extract are found in the complex. A free pool of Cdc45 and GINS proteins may be required for activation of replication origins throughout S phase. Only a small percentage of Mcm proteins in the nucleus are in the CMG complex; therefore, most Mcm proteins may not be located at sites of DNA unwinding. The vast excess of Mcm2-7 in the extracts that is not in the CMG complex is consistent with reports that most Mcm proteins do not localize to sites of DNA replication in metazoans (Moyer, 2006).

Taking further the notion that the helicase activity of the CMG complex is provided by Mcm2-7, what role might Cdc45 and GINS play in this function? It is possible that the CMG complex forms only at preinitiation sites and that, concomitant with this assembly, a specific set of posttranslational modifications of the initiation factors activates the helicase activity. Further dissection of the modification patterns of the proteins of the CMG complex and an understanding of how the complex is assembled will answer these questions. Apart from specific modifications, a simple notion would be that Cdc45 and/or GINS induces or stabilizes a conformational change in the Mcm2-7 hexamer, serving as a molecular switch that converts an inactive helicase to an active form. Cdc45 and GINS may thus be a part of the actual helicase machinery, with, for example, Cdc45 possibly serving as a wedge in the recently proposed 'plowshare' model for helicase activity. A second, nonexclusive possibility is that Cdc45 and GINS associate with the Mcm2-7 helicase complex for purposes of coordination with DNA repair factors. Cdc45 has been shown to associate with the checkpoint proteins Mrc1 and Tof1 during S phase, and it is stressed that the stringent purification methods might dislodge the Drosophila homolog of Claspin/Mrc1 from the CMG complex. The cell's central replicative helicase may only be activated when the factors that can coordinate helicase function with checkpoint proteins are fully engaged (Moyer, 2006).

The data presented in this study indicate that a purified Mcm2-7-containing complex has DNA helicase activity. The question may be raised of whether the Mcm2-7 proteins of the CMG complex are functioning as a helicase or whether the CMG complex either dissociates to a Mcm4,6,7 subcomplex or copurifies with an unrecognized helicase (Moyer, 2006).

Although neither of these possibilities can be formally disproven, the data suggest that both of these possibilities are unlikely. The CMG complex is stable through many chromatography steps and high-salt conditions, and it is believed that it is unlikely that the complex would dissociate during the gentle conditions of the helicase assays. In addition, if it did dissociate, then the free Mcm2, Mcm3, and Mcm5 that would result from the dissociation would be expected to inhibit the Mcm4,6,7 activity. The second possibility, that there is an unrecognized helicase complex that copurifies with the CMG complex, is also unlikely. Depletion experiments indicate that the observed helicase activity is tightly associated with the CMG complex. Examination of the CMG complex purified by anti-Cdc45, anti-FLAG/anti-Cdc45, or anti-Psf2 chromatography suggests that CMG does not have any unidentified binding partners that could provide helicase activity. Hence, the best explanation of the data shown in this study is that the observed helicase activity is manifest from the complex itself (Moyer, 2006).

Electron microscope images of Mcm2-7 and GINS complexes individually have shown that both complexes form ring-shaped structures. These findings suggest that the two rings may stack on top of each other to form a common central channel that could surround ssDNA or dsDNA. A speculative model of the molecular architecture of the CMG complex is presented. Double hexamers of the archaeal Mcm rings have been observed, and, by analogy, one might expect the Drosophila Mcm2-7 to form such double hexamer structures. However, the present model posits that the active helicase contains a six-member (Mcm2-7) and four-member (GINS) ring ensemble. The model of one Mcm2-7 hexamer per CMG complex is based on gel filtration data, which show that the CMG complex migrates at the same position as thyrogloblulin (669 kDa) on both Superdex-200 and Superose 6 columns, suggesting that the mass of the CMG complex is close to 700 kDa. The calculated molecular mass of a complex containing one Mcm2-7 hexamer, one GINS tetramer, and one Cdc45 molecule is 708 kDa (Moyer, 2006).

It has been shown that Mcm2, Mcm5, Mcm7, Sld5, and Cdc45 localize to sites of DNA unwinding on a plasmid in Xenopus extracts, a result that is consistent with the present data. In that study, unwinding was uncoupled from DNA synthesis, and some fraction of DNA replication fork proteins were associated with the unwinding activity. These researchers suggested that the entire set of Mcm2-7, Cdc45, and the GINS complex are part of a molecular machine that is referred to as the 'unwindosome.' The term unwindosome may perhaps refer to a very large collection of proteins yet to be identified that comprise and associate with the DNA unwinding machinery. In fact, another study shows that a large number of S. cerevisiae proteins associate with Sld5 and Mcm4 in a high-molecular-weight complex referred to as the 'replisome progression complex.' This study has shown that Cdc45, Mcm2-7, and GINS form a biochemically discrete complex, and it is proposed that the CMG complex per se forms the core of helicase activity (Moyer, 2006).

Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. This report shows that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and it is suggested that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage (Ilves, 2012).

disc proliferation abnormal: Biological Overview | Evolutionary Homologs | Developmental Biology | References

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