Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress.
The regulation of a pre-replicative complex (pre-RC) at origins ensures that the genome is replicated only once per cell cycle. Cdt1 is an essential component of the pre-RC that is rapidly degraded at G1-S and also inhibited by Geminin (Gem) protein to prevent re-replication. We have previously shown that destruction of the Drosophila homolog of Cdt1, Double-parked (Dup), at G1-S is dependent upon cyclin-E/CDK2 and important to prevent re-replication and cell death. Dup is phosphorylated by cyclin-E/Cdk2, but this direct phosphorylation was not sufficient to explain the rapid destruction of Dup at G1-S. Here, we present evidence that it is DNA replication itself that triggers rapid Dup destruction. We find that a range of defects in DNA replication stabilize Dup protein and that this stabilization is not dependent on ATM/ATR checkpoint kinases. This response to replication stress was cell-type specific, with neuroblast stem cells of the larval brain having the largest increase in Dup protein. Defects at different steps in replication also increased Dup protein during an S-phase-like amplification cell cycle in the ovary, suggesting that Dup stabilization is sensitive to DNA replication and not an indirect consequence of a cell-cycle arrest. Finally, we find that cells with high levels of Dup also have elevated levels of Gem protein. We propose that, in cycling cells, Dup destruction is coupled to DNA replication and that increased levels of Gem balance elevated Dup levels to prevent pre-RC reformation when Dup degradation fails.
May
Noah R
NR
Department of Genetics, University of Pennsylvania School of Medicine, 415 Curie Blvd, Philadelphia, PA 19104-6145, USA.
Thomer
Marguerite
M
Murnen
Katherine F
KF
Calvi
Brian R
BR
D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D005260:Female; D016195:G2 Phase / Q000502:physiology; D064151:Minichromosome Maintenance Complex Component 6; D009154:Mutation; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C411983:DUP protein, Drosophila; D029721:Drosophila Proteins; C491204:Mcm6 protein, Drosophila; D064151:Minichromosome Maintenance Complex Component 6