latheo Encodes a Subunit of the Origin Recognition Complex and Disrupts Neuronal Proliferation and Adult Olfactory Memory When Mutant

Pinto, Shirly; Quintana, David G.; Smith, Patrick; Mihalek, Robert; Hou, Zhi-hui; Boynton, S; Jones, Christopher J.; Hendricks, Michael; Velinzon, Klara; Wohlschlegel, James A.; Austin, Robert J.; Lane, William S.; Tully, Tim; Dutta, Anindya

doi:10.1016/s0896-6273(00)80752-7

Cited by 106 publications

(89 citation statements)

References 51 publications

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“…However, the direct molecular mechanisms governing the control of ORC function following Cdk phosphorylation has not been previously examined. While the data presented in this study are discussed in light of the role of DmORC in the initiation of DNA replication, it has to be stressed that DmORC and its individual subunits also have functions in cytokinesis, heterochromatin formation, and at the neuromuscular junction, which are distinct and separable from their replication functions (55)(56)(57)(58). We therefore anticipate that DmORC phosphorylation would also modulate activity of these other functions.…”

Section: Discussionmentioning

confidence: 92%

CDK Phosphorylation Inhibits the DNA-binding and ATP-hydrolysis Activities of the Drosophila Origin Recognition Complex

Remus¹,

Blanchette²,

Rio

et al. 2005

Journal of Biological Chemistry

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Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2⅐cyclin E and Cdk1⅐cyclin B in vitro. Such phosphorylation of DmORC by Cdk2⅐cyclin E, but not by Cdk1⅐cyclin B, requires an "RXL" motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.Prevention of re-replication in eukaryotic cells is essential for the maintenance of genomic ploidy, and mechanisms preventing such reinitiation-associated events are needed because unscheduled or uncontrolled DNA replication initiation can lead to genomic instability (1-5). Re-replication control is achieved through (i) the prevention of repeated origin firing during one S phase and (ii) coordination of chromosomal replication with the segregation of the sister chromatids in mitosis. Genetic studies in the fission yeast Schizosaccharomyces pombe first demonstrated that cyclin-dependent kinases (Cdks) 4 play a prominent role in preventing re-replication during S phase and coupling S phase progression to chromosomal segregation in mitosis, by showing that inhibition of Cdk activity in G 2 /M can induce re-entry into S phase in the absence of mitosis (6 -8). Additional studies in the budding yeast, Saccharomyces cerevisiae, substantiated these findings and demonstrated that some Cdk activity is required for one round of replication and that higher levels accumulating in S phase prevent re-initiation. Thus Cdks have both negative and positive roles in the initiation of DNA replication (9). It is now known that in all eukaryotes studied Cdks contribute to the prevention of re-replication through direct inactivation of pre-RC proteins via multiple redundant mechanisms. This redundancy ensures that deregulation of any single mechanism is not sufficient to induce genomic re-replica...

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Section: Discussionmentioning

confidence: 92%

CDK Phosphorylation Inhibits the DNA-binding and ATP-hydrolysis Activities of the Drosophila Origin Recognition Complex

Remus¹,

Blanchette²,

Rio

et al. 2005

Journal of Biological Chemistry

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“…Mutants, homozygous for ORC2, ORC3, or ORC5, all die in larval stages as large maternal ORC stores are depleted. In the terminal stages, there is a dramatic decrease in DNA replication and cellular proliferation (20)(21)(22)(23). Furthermore, females harboring hypomorphic mutations in ORC2 are sterile because they do not amplify the chorion genes in follicle cells, where ORC is localized at four discrete amplification foci (24)(25)(26).…”

mentioning

confidence: 99%

Functional analysis of mutant and wild-type Drosophila origin recognition complex

Chesnokov

Remus

Botchan

2001

Proc. Natl. Acad. Sci. U.S.A.

112

150

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The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wildtype and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA ؉ nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.Drosophila ORC ͉ DNA replication ͉ ATPase activity ͉ DNA binding T he initiation of DNA replication in higher eukaryotes occurs at thousands of sites along chromosomes, and significant progress has been made in understanding how proteins work to regulate this event such that each region replicates exactly once per cell cycle (1). The origin recognition complex (ORC), a heteromeric six-subunit protein, is a central component for DNA replication. ORC binds to DNA at replication origin sites and serves in some way as a scaffold for assembly of other key initiation factors such as cdc6, cdt1, MCM complex, and cdc45 (2). How these factors eventually lead to a melting of the duplex strands and an engagement of the DNA polymerase alpha is still unknown. A deeper understanding of how the cell cycle machinery triggers initiation and couples this event to signal transduction pathways requires a finer dissection of the initiation process. The initiator role of ORC in the DNA replication process was first discovered in Saccharomyces cerevisiae (3), where special sequences of about 100 base pairs serve as origin (ori) sites (4). Within these elements, a short 11-base pair consensus sequence serves as a core binding site for ORC.Although there is a gratifying conservation of such protein factors in different organisms, a surprising divergence for the cis-acting origin sequences almost certainly underscores evolutionary changes in the way DNA replication is regulated and the addition of roles for replication in sculpting functions of the chromosome. For example, in Saccharomyces pombe, ori sequences appear to be largely asymmetric stretches of AT that do not show consensus sequence elements. DNA binding by ORC may depend on a special N-terminal domain of the ORC4 subunit that contains multiple A͞T hook motifs that can each recognize such DNA ele...

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“…Similarly, in Drosophila, a conditional mutation in the DmORC2 gene demonstrated strong effects (16). In human cells, putative ORC genes have also been identified (15,(17)(18)(19)(20)(21)(22)(23), and interaction of their products has been reported (17, 21-23), suggesting that a similar protein complex might be functional during DNA replication in human cells.Recent studies have demonstrated that several events in nuclei, including DNA replication, take place in specialized compartments, in which specific factors assemble in highly organized structures. It is essential to ascertain the distribution of replication proteins among subcelluar fractions and their interactions to understand how DNA replication is initiated and otherwise regulated.…”

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confidence: 99%

mentioning

confidence: 99%

Association of Human Origin Recognition Complex 1 with Chromatin DNA and Nuclease-resistant Nuclear Structures

Tatsumi

Tsurimoto

Shirahige

et al. 2000

Journal of Biological Chemistry

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An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.The replication of eukaryotic chromosome occurs in a highly regulated manner during the S phase. It is important to understand coordinated action of various proteins acting on specific cis-elements during initiation of chromosomal replication. In budding yeast (Saccharomyces cerevisiae), the origin recognition complex (ORC), 1 composed of six polypeptides, is essential for initiation of DNA replication, binding specifically to replication origins throughout the cell cycle (1-6). Prior to the initiation of DNA replication, ORC forms a large protein complex, called a prereplicative complex, by association with other initiation factors, including CDC6 and MCM proteins. After the initiation of DNA replication, the prereplicative complex change to the postreplicative form as indicated by dissociation of factors from ORC in parallel with alteration of the ORC-DNA interaction (3,(5)(6)(7)(8). Thus, dynamic change of DNA-protein complexes at replication origins in S phase appears to be closely connected with the initiation of DNA replication.The counterparts of prereplicative complex components, such as ORC, CDC6, and MCMs, have been identified in various eukaryotes. Thus, the basic mechanisms for initiation of DNA replication seem to be highly conserved. Putative ORC subunits constituting similar multiprotein complexes as that in S. cerevisiae have been identified in Drosophila (9, 10) and Xenopus (11-15). In the latter, depletion of ORC from egg extracts results in inhibition of the initiation of DNA replication (11)(12)(13)(14). Similarly, in Drosophila, a conditional mutation in the DmORC2 gene demonstrated strong effects (16). In human cells, putative ORC genes have also been identified (15,(17)(18)(19)(20)(21)(22)(23), and interaction of ...

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latheo Encodes a Subunit of the Origin Recognition Complex and Disrupts Neuronal Proliferation and Adult Olfactory Memory When Mutant

Cited by 106 publications

References 51 publications

CDK Phosphorylation Inhibits the DNA-binding and ATP-hydrolysis Activities of the Drosophila Origin Recognition Complex

CDK Phosphorylation Inhibits the DNA-binding and ATP-hydrolysis Activities of the Drosophila Origin Recognition Complex

Functional analysis of mutant and wild-type Drosophila origin recognition complex

Association of Human Origin Recognition Complex 1 with Chromatin DNA and Nuclease-resistant Nuclear Structures

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