The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S. cerevisiae. Origin recognition by ORC requires ATP. Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP. ATP binding and hydrolysis by Orc1p are both regulated by origin DNA in a sequence-specific manner. ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC-origin interaction, indicating that ATP is a cofactor that locks ORC on origin DNA. These data demonstrate that occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC.

We isolated mutations in Drosophila E2F and DP that affect chorion gene amplification and ORC2 localization in the follicle cells. In the follicle cells of the ovary, 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.

Hepsin is a membrane-anchored, trypsin-like serine protease with prominent expression in the human liver and tumours of the prostate and ovaries. To better understand the biological functions of hepsin, we identified macromolecular substrates employing a tetrapeptide PS-SCL (positional scanning-synthetic combinatorial library) screen that rapidly determines the P1-P4 substrate specificity. Hepsin exhibited strong preference at the P1 position for arginine over lysine, and favoured threonine, leucine or asparagine at the P2, glutamine or lysine at the P3, and proline or lysine at the P4 position. The relative activity of hepsin toward individual AMC (7-amino-4-methylcoumarin)-tetrapeptides was generally consistent with the overall peptide profiling results derived from the PC-SCL screen. The most active tetrapeptide substrate Ac (acetyl)-KQLR-AMC matched with the activation cleavage site of the hepatocyte growth factor precursor sc-HGF (single-chain HGF), KQLR downward arrowVVNG (where downward arrow denotes the cleavage site), as identified by a database analysis of trypsin-like precursors. X-ray crystallographic studies with KQLR chloromethylketone showed that the KQLR peptide fits well into the substrate-binding cleft of hepsin. This hepsin-processed HGF induced c-Met receptor tyrosine phosphorylation in SKOV-3 ovarian cancer cells, indicating that the hepsin-cleaved HGF is biologically active. Activation cleavage site mutants of sc-HGF with predicted non-preferred sequences, DPGR downward arrowVVNG or KQLQ downward arrowVVNG, were not processed, illustrating that the P4-P1 residues can be important determinants for substrate specificity. In addition to finding macromolecular hepsin substrates, the extracellular inhibitors of the HGF activator, HAI-1 and HAI-2, were potent inhibitors of hepsin activity (IC50 4+/-0.2 nM and 12+/-0.5 nM respectively). Together, our findings suggest that the HGF precursor is a potential in vivo substrate for hepsin in tumours, where hepsin expression is dysregulated and may influence tumorigenesis through inappropriate activation and/or regulation of HGF receptor (c-Met) functions.

We found that PPM1D, encoding a serine/threonine protein phosphatase, lies within an epicenter of the region at 17q23 that is amplified in breast cancer. We show that overexpression of this gene confers two oncogenic phenotypes on cells in culture: attenuation of apoptosis induced by serum starvation and transformation of primary cells in cooperation with RAS.

Cyclin-dependent kinases are critical regulators of eukaryotic DNA replication. We show that the S-phase cyclin Clb5 binds stably and directly to the origin recognition complex (ORC). This interaction is mediated by an "RXL" target sequence, or "Cy" motif, in the Orc6 subunit that is recognized by the "hydrophobic patch" region on Clb5. The Clb5-Orc6 interaction requires replication initiation, and is maintained throughout the remainder of S phase and into M phase. Eliminating the Clb5-Orc6 interaction has no effect on initiation of replication but instead sensitizes cells to lethal overreplication. We propose that Clb5 binding to ORC provides an origin-localized replication control switch that specifically prevents reinitiation at replicated origins.[Keywords: Cell cycle control; DNA replication; cyclin-dependent kinase; origin recognition complex; genomic stability; re-replication] Supplemental material is available at http://www.genesdev.org. The eukaryotic cell cycle is controlled by oscillations in cyclin-dependent kinase (Cdk) activity (Zachariae and Nasmyth 1999). Cdk activity oscillations are required because critical cell cycle steps are both positively and negatively regulated by Cdk activity. A well-characterized example is DNA replication (Bell and Dutta 2002), in which oscillating Cdk levels control the formation and activation of protein complexes at origins of DNA replication.In budding yeast, origins are bound throughout the cell cycle by the six-member origin recognition complex (ORC;Diffley et al. 1994). As cells pass from the M to the G1 phase of the cell cycle, Cdc6 and Cdt1 proteins interact with the ORC-bound origin and direct the loading of the six-member Mcm2-7 complex (Cocker et al. 1996;Aparicio et al. 1997;Tanaka et al. 1997;Devault et al. 2002;Tanaka and Diffley 2002). The resulting structure is called the "pre-Replicative Complex" or pre-RC.As cells enter S phase, elevated levels of Cdk activity stimulate initiation of DNA replication from the pre-RC loaded origins (Schwob et al. 1994). Positive control of replication by Cdk activity in Saccharomyces cerevisiae is primarily mediated by S-phase cyclins Clb5 and Clb6 (Schwob and Nasmyth 1993;Schwob et al. 1994;Donaldson et al. 1998a;Donaldson 2000; Epstein and Cross 2002), although it is not well understood how Cdk activity induces replication. Cdk phosphorylation of the Sld2/Drc1 replication protein has been demonstrated to be essential for replication initiation (Masumoto et al. 2002), but there may be additional activating substrates. During S phase, ORC is normally phosphorylated by Clb5,6, and biochemical studies have identified an interaction between Clb5 and ORC (Weinreich et al. 2001). The function of this interaction has been unclear. In Xenopus, cyclin E is recruited to origins of replication through Cdc6 to activate replication (Furstenthal et al. 2001a,b), although the targets for cyclin E-directed Cdk activity are also unclear.

In the yeast Saccharomyces cerevisiae, sequence-specific DNA binding by the origin recognition complex (ORC) is responsible for selecting origins of DNA replication. In metazoans, origin selection is poorly understood and it is unknown whether specific DNA binding by metazoan ORC controls replication. To address this problem, we used in vivo and in vitro approaches to demonstrate that Drosophila ORC (DmORC) binds to replication elements that direct repeated initiation of replication to amplify the Drosophila chorion gene loci in the follicle cells of egg chambers. Using immunolocalization, we observe that ACE3, a 440-bp chorion element that contains information sufficient to drive amplification, directs DmORC localization in follicle cells. Similarly, in vivo cross-linking and chromatin immunoprecipitation assays demonstrate association of DmORC with both ACE3 and two other amplification control elements, AER-d and ACE1. To demonstrate that the in vivo localization of DmORC is related to its DNA-binding properties, we find that purified DmORC binds to ACE3 and AER-d in vitro, and like its S. cerevisiae counterpart, this binding is dependent on ATP. Our findings suggest that sequence-specific DNA binding by ORC regulates initiation of metazoan DNA replication. Furthermore, adaptation of this experimental approach will allow for the identification of additional metazoan ORC DNA-binding sites and potentially origins of replication.

The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.