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: 4.6). 01/2006; 280(48):39740-51. DOI: 10.1074/jbc.M508515200
Source: PubMed

ABSTRACT 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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    ABSTRACT: Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.
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    ABSTRACT: Every time a cell divides it must faithfully duplicate its genome before the cell divides. If replication initiates a second time (re-replication) before cytokinesis, cells can accumulate extensive DNA damage, which results in genomic instability, a hallmark of tumorigenesis. To prevent re-replication eukaryotic cells must inhibit the re-initiation of replication start sites, or origins, across the genome. Examples of both Cyclin-Dependent Kinase (CDK)-dependent and CDK-independent mechanisms have been identified that regulate the components of the pre-Replicative Complex (pre-RC) to prevent re-replication. The pre-RC is a multi-protein complex that assembles at origins during G1, before DNA replication begins. After an origin initiates pre-RC components must be prevented from reassembling at origins until the next cell cycle. When the mechanisms preventing re-replication in the yeast Saccharomyces cerevisiae are disrupted, unregulated replication occurs. Not all origins are capable of re-initiating during this re-replication. Rather, a subset of all potential origin sequences reform pre-RCs, and of those, only a portion re-initiates. The origins that re-initiate do not correlate with any other known subclass of origins (e.g. - early/late initiating origins). (cont.) The inability of some origins to form pre-RCs during re-replication might be due to restrictive chromatin structure preventing pre-RC components from associating with origin DNA. Similarly, origins that form pre-RCs but do not re-initiate might be prevented from recruiting replication machinery due to a restrictive chromatin structure. In addition, these origins might not re-initiate because replication factors that function downstream of pre-RC components also could be regulated to prevent re-replication. One of the mechanisms that S. cerevisiae and other eukaryotes use to prevent re-replication is phosphorylating one or multiple subunits of the Origin Recognition Complex (ORC). In S. cerevisiae, Orc2 and Orc6 are both phosphorylated but have distinct mechanisms for preventing re-replication. Phosphorylating Orc2 results in the direct inhibition of pre-RC assembly whereas phosphorylating Orc6 helps stabilize CDK at origins. By contrast, of CDK helps to prevent re-replication, most likely through a combination of catalytic activity and steric hindrance. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006. Includes bibliographical references.