In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila melanogaster embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase–promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development. Author summary Cells divide rapidly in the early embryos of most animals. However, during a conserved period of development known as the mid-blastula transition (MBT), the cell cycle slows down dramatically. In Drosophila embryos, genome duplication abruptly slows to initiate this cell cycle prolongation. This is achieved through the onset of late replication, a well-recognized phenomenon in which specific sequences of the genome await replication until long after other sequences have finished. Even though this temporal program of replication is a major determinant of the duration of S phase, the factors involved in this process remain unknown. Here, we use genetics and real-time microscopy to visualize replication in developing fly embryos and show that the protein Rap1 interacting factor 1 (Rif1) mediates the introduction of late replication at the MBT. We find that at this stage, Rif1 binds to and selectively delays the replication of large blocks of repetitive DNA known as satellite sequences. During the rapid cell cycles before the MBT, we show that the cyclin-dependent kinase 1 (Cdk1) prevents Rif1 from slowing down DNA replication by driving its removal from the chromatin. The developmental down-regulation of Cdk1 at the MBT allows Rif1 to associate with the satellite sequences and initiate cell cycle slowing. Our work provides new insights into the temporal programming of S phase and into the embryonic origin of late replication.
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