4807 Introduction During every cell cycle the entire genome must be copied, but it is important that each region replicates only once to maintain genome integrity. To prevent over-replication, the activity of origins of replication is tightly controlled. Beginning in late mitosis, origins are prepared for replication by binding of a pre- replicative complex (pre-RC), which is subsequently activated to initiate replication at the onset of S phase (reviewed by Bell and Stillman, 1992) (Bell and Dutta, 2002; Diffley et al., 1994). The building of the pre-RC onto origins in late mitosis/early G1 is a stepwise process. The origin recognition complex (ORC) serves as a scaffold for subsequent association of Cdc6 and Cdt1, both of which are required to load the Minichromosome Maintenance (MCM) complex, the replicative helicase (Bell and Stillman, 1992; Cocker et al., 1996; Diffley et al., 1994) (reviewed by Diffley, 2001) (Liang et al., 1995; Maiorano et al., 2000; Nishitani et al., 2000; Nishitani and Nurse, 1995; Tada et al., 2001). Once MCMs are loaded, the origin is considered to be licensed for subsequent replication (Chong et al., 1995). Cdc7 kinase, with its activating subunit Dbf4, and CDK2 kinase, activated by cyclin E or cyclin A, are then required for initiation of replication (reviewed by Hengstschlager et al., 1999; Sclafani, 2000) (Dowell et al., 1994; Lei et al., 1997; Masumoto et al., 2002; Zou and Stillman, 1998). Initiation is associated with departure of Cdc6, Cdt1, MCMs, and, in multicellular eukaryotes, certain ORC subunits from the origin (reviewed by Bell and Dutta, 2002). Continued CDK activity in S, G2 , and early M phases inhibits reassembly of the pre-RC to block origin refiring (Dahmann et al., 1995; Hayles et al., 1994). Unique to multicellular eukaryotes is another inhibitor of pre-RC assembly, Geminin, which binds Cdt1 and renders it incapable of loading the MCM complex (McGarry and Kirschner, 1998; Quinn et al., 2001; Tada et al., 2001; Wohlschlegel et al., 2000). It is only after Geminin and cyclins are degraded at the subsequent metaphase that the pre-RC can reform, thereby restricting origin licensing, and DNA replication, to once per segregation of chromosomes (Dahmann et al., 1995; Diffley et al., 1994; Noton and Diffley, 2000; Piatti et al., 1996; Tada et al., 2001). Although phosphorylation of pre-RC subunits appears to be important for initiation and to block pre-RC re-assembly, the biochemical mechanisms are not fully understood. In the yeasts Saccharomyces cerevisiae and S. pombe, CDKs block re- replication by phosphorylating several pre-RC targets including CDC6 and subunits of the ORC and MCM complex (Drury et al., 2000; Gopalakrishnan et al., 2001; Jallepalli et It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G 2 phase. We have investigated the developmental regulation of Double- parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms. We find that phosphorylation and degradation of Dup protein at G 1 /S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound re- replication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, our results indicate that stringent regulation of Dup protein is critical to protect genome integrity. Key words: DNA replication, Cyclin E, Double-parked, Cdt1, Chorion gene amplification Summary Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2 Marguerite Thomer*, Noah R. May*, Bhagwan D. Aggarwal, Garrick Kwok and Brian R. Calvi † Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145, USA *These authors contributed equally to this work † Author for correspondence (e-mail: calvi@mail.med.upenn.edu) Accepted 5 July 2004 Development 131, 4807-4818 Published by The Company of Biologists 2004 doi:10.1242/dev.01348 Research article