Drosophila double-parkedis sufficient to induce re-replication during development and is regulated by cyclin E/CDK2

Abstract

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 G2 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 G1/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 rereplication 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.

Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress

“…To test this idea, we used a heat-inducible hsp70:Myc:FL-Dup strain to examine the effect of Dup overexpression on mitotic cycle and endocycling follicle cells. Expression was induced by three 30-min heat pulses over 30 h, as we described previously (Thomer et al 2004). In a subset of both mitotic cycle and endocycling follicle cells, Dup overexpression resulted in enlarged nuclei, an increase in total fluorescent intensity after labeling with the DNA dyes DAPI or TOTO-3, and many incorporated BrdU, suggesting that they were actively rereplicating to a higher DNA copy number ( Fig.…”

“…1B; Thomer et al 2004). To investigate whether this cell death is indeed apoptosis, we labeled with several markers.…”

“…We had previously shown that Dup protein is phosphorylated by Cyclin E/CDK2 and targeted for proteolysis at G1/S (Thomer et al 2004;May et al 2005). We showed that this proteolysis is important because even small increases in Dup protein levels were sufficient to cause rereplication.…”

“…Thereafter, they selectively rereplicate only a few loci, resulting in amplification of gene copy number (Calvi et al 1998). Similar to mitotic cycles, MCM proteins periodically associate with chromatin during each endocycle G phase, and Dup protein is proteolyzed at the G/S transition, suggesting that Dup regulation may be important for licensing control of origins in the endocycle (Su and O'Farrell 1998;Whittaker et al 2000;Thomer et al 2004;Hong et al 2007). To test this idea, we used a heat-inducible hsp70:Myc:FL-Dup strain to examine the effect of Dup overexpression on mitotic cycle and endocycling follicle cells.…”

An ATR- and BRCA1-Mediated Fanconi Anemia Pathway Is Required for Activating the G2/M Checkpoint and DNA Damage Repair upon Rereplication

“…Cdt1 is a replication initiation factor that promotes the assembly of the prereplicative complex (20,28). Overexpression of Cdt1 in human cells results in rereplication (43), and increased expression of Drosophila melanogaster Cdt1 (Dup) in diploid cells is sufficient to induce polyploidization (40). In Xenopus laevis egg extract, addition of Cdt1 after one round of DNA replication causes rereplication (2).…”