CDK phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of the Drosophila origin recognition complex

Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3204, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2006; 280(48):39740-51. DOI: 10.1074/jbc.M508515200
Source: PubMed


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 Cdk2 x cyclin E and Cdk1 x cyclin B in vitro. Such phosphorylation of DmORC by Cdk2 x cyclin E, but not by Cdk1 x cyclin 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.

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    • "Only the Orc1, Orc2, and Orc6 subunits contain potential CDK phosphorylation sites, and CDKdependent phosphorylation of Orc2 destabilizes the remaining ORC subunits (Lee et al., 2012b). This S phase dependent release of ORC(1–6) from chromatin occurs also in flies, frogs, and worms (Rowles et al., 1999; Sun et al., 2002; Remus et al., 2005; Sonneville et al., 2012 "
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    ABSTRACT: Development of a fertilized human egg into an average sized adult requires about 29 trillion cell divisions, thereby producing enough DNA to stretch to the Sun and back 200 times (DePamphilis and Bell, 2011)! Even more amazing is the fact that throughout these mitotic cell cycles, the human genome is duplicated once and only once each time a cell divides. If a cell accidentally begins to re-replicate its nuclear DNA prior to cell division, checkpoint pathways trigger apoptosis. And yet, some cells are developmentally programmed to respond to environmental cues by switching from mitotic cell cycles to endocycles, a process in which multiple S phases occur in the absence of either mitosis or cytokinesis. Endocycles allow production of viable, differentiated, polyploid cells that no longer proliferate. What is surprising is that among the 516 (Manning et al., 2002) to 557 (BioMart web site) protein kinases encoded by the human genome, only eight regulate nuclear DNA replication directly. These are Cdk1, Cdk2, Cdk4, Cdk6, Cdk7, Cdc7, Checkpoint kinase-1 (Chk1), and Checkpoint kinase-2. Even more remarkable is the fact that only four of these enzymes (Cdk1, Cdk7, Cdc7, and Chk1) are essential for mammalian development. Here we describe how these protein kinases determine when DNA replication occurs during mitotic cell cycles, how mammalian cells switch from mitotic cell cycles to endocycles, and how cancer cells can be selectively targeted for destruction by inducing them to begin a second S phase before mitosis is complete.
    Frontiers in Physiology 09/2012; 3:368. DOI:10.3389/fphys.2012.00368 · 3.53 Impact Factor
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    • "This model is also consistent with observations made in other systems, for example, in CHO hamster cells where Cdk1/CycA was able to hyper-phosphorylate ORC1 and decrease its affinity for chromatin (Li et al., 2004). Moreover, it has also been shown that Cdk1/CyclinA can phosphorylate ORC2 in vitro (Remus et al., 2005). All these data support a model where CycA, in a dosedependent manner, promotes ORC2 shuttling between euchromatin and heterochromatin as a mechanism to accomplish the early S phase (Fig. 7D). "
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    ABSTRACT: Endocycles, which are characterised by repeated rounds of DNA replication without intervening mitosis, are involved in developmental processes associated with an increase in metabolic cell activity and are part of terminal differentiation. Endocycles are currently viewed as a restriction of the canonical cell cycle. As such, mitotic cyclins have been omitted from the endocycle mechanism and their role in this process has not been specifically analysed. In order to study such a role, we focused on CycA, which has been described to function exclusively during mitosis in Drosophila. Using developing mechanosensory organs as model system and PCNA::GFP to follow endocycle dynamics, we show that (1) CycA proteins accumulate during the last period of endoreplication, (2) both CycA loss and gain of function induce changes in endoreplication dynamics and reduce the number of endocycles, and (3) heterochromatin localisation of ORC2, a member of the Pre-RC complex, depends on CycA. These results show for the first time that CycA is involved in endocycle dynamics in Drosophila. As such, CycA controls the final ploidy that cells reached during terminal differentiation. Furthermore, our data suggest that the control of endocycles by CycA involves the subnuclear relocalisation of pre-RC complex members. Our work therefore sheds new light on the mechanism underlying endocycles, implicating a process that involves remodelling of the entire cell cycle network rather than simply a restriction of the canonical cell cycle.
    Development 02/2012; 139(3):547-57. DOI:10.1242/dev.069823 · 6.46 Impact Factor
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    • "This activation marks the end of origin licensing and the start of origin firing (Labib & Diffley 2001). Studies in several organisms have shown that the onset of S-phase requires CDK (cyclin dependent kinase) and DDK (Dbf-4 dependent Cdc7 kinase) activity to promote activation of the MCM2-7 helicase, while at the same time the recruitment of pre-replication complexes is inhibited (Bousset & Diffley 1998; Nguyen, et al. 2001; Remus, et al. 2005). CDKs and DDK4 are not only required for the activation of the MCM complex, they also trigger the assembly of additional factors. "

    DNA Replication and Related Cellular Processes, 09/2011; , ISBN: 978-953-307-775-8
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