Rif1 restrains the rate of replication origin firing in Xenopus laevis.

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles.

Polo-like kinase 1 (Plk1) regulates DNA replication origin firing and interacts with Rif1 in Xenopus.

The activation of eukaryotic DNA replication origins needs to be strictly controlled at multiple steps in order to faithfully duplicate the genome and to maintain its stability. How the checkpoint recovery and adaptation protein Polo-like kinase 1 (Plk1) regulates the firing of replication origins during non-challenged S phase remained an open question. Using DNA fiber analysis, we show that immunodepletion of Plk1 in the Xenopus in vitro system decreases replication fork density and initiation frequency.

Organization of DNA Replication Origin Firing in Egg Extracts: The Role of Intra-S Checkpoint.

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint.

Polo-like kinase 1 (Plk1) is a positive regulator of DNA replication in the system.

Polo-like kinase 1 (Plk1) is a cell cycle kinase essential for mitosis progression, but also important for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is expressed in S phase, little is known about its function during unperturbed DNA replication. Using egg extracts, mimicking early embryonic replication, we demonstrate that Plk1 is simultaneously recruited to chromatin with pre-replication proteins where it accumulates throughout S phase.

Genome wide decrease of DNA replication eye density at the midblastula transition of .

During the first rapid divisions of early development in many species, the DNA:cytoplasm ratio increases until the midblastula transition (MBT) when transcription resumes and cell cycles lengthen. S phase is very rapid in early embryos, about 20-30 times faster than in differentiated cells. Using a combination of DNA fiber studies and a embryonic replication system, we show that S phase slows down shortly after the MBT owing to a genome wide decrease of replication eye density.

On the Interplay of the DNA Replication Program and the Intra-S Phase Checkpoint Pathway.

DNA replication in eukaryotes is achieved by the activation of multiple replication origins which needs to be precisely coordinated in space and time. This spatio-temporal replication program is regulated by many factors to maintain genome stability, which is frequently threatened through stresses of exogenous or endogenous origin. Intra-S phase checkpoints monitor the integrity of DNA synthesis and are activated when replication forks are stalled.