Dynamics of DNA Replication in Higher Eukaryotes
In higher eukaryotes DNA replication starts at thousands of sites in the genome called replication origins. Our group is interested how the activation of these multiple origins is coordinated in space and time and how this is linked to genomic instability which leads to cancer.
Eukaryotic DNA replication requires a strict control during S phase in order to maintain genome stability. In higher eukaryotes the DNA replication starts from several thousand replication origins which are activated at different time during the S phase (Figure 1). Little is known about the molecular mechanisms that regulate the spatio-temporal program of replication origins in higher eukaryotes. Defects in the replication program provokes genomic instability and cancer. The replication program is closely linked to the replication checkpoint which protects the integrity of the genome. In response to stalled replication forks and DNA breaks the essential checkpoint proteins ATR and Chk1 are activated which leads to the inhibition of late firing replication clusters.
The spatio-temporal replication program in Xenopus during development
In order to study the replication program in higher eukaryotes we use the Xenopus model system which allowed in the past to identify a great number of replication and checkpoint factors. This in vitro system, which consists of isolated sperm nuclei replicating efficiently in egg extracts, mimics early embryonic cell cycles with very short S phases. During the first rapid divisions in Xenopus embryos transcription is repressed and the ratio of nuclei to cytoplasm (nuclear-cytoplasmic ratio) increases therefore progressively. At the midblastula transition (MBT) between the 12.-13. division, the S phase lengthens, the G1 and G2 phases of the cell cycle are introduced and widespread zygotic transcription initiates. S phase in differentiated cells takes several hours. It has been proposed that the spatial and presumably also the temporal replication pattern changes at the mid-blastula transition in Xenopus. In the Xenopus in vitro system where S phase lasts about 30 min we and others have shown that replication origins are spaced 5-15 kb and are clustered in early and late firing groups of origins (Marheineke and Hyrien 2001, Marheineke and Hyrien 2004). It has also been shown that several replication factors are titrated by the increase of the nucleo-cytosolic ratio after 12 divisions during early development which triggers the DNA replication checkpoint, slows down S phase and which would trigger the mid-blastula transition (MBT). Our lab investigates the mechanisms of the replication program in this developmental context. We have recently shown that shortly after the MBT the number of replication eyes decreases genome wide about 30% (Platel et al, 2019) (figure 2).
We use the DNA combing technique, a DNA fiber stretching technique in order to analyse the replication program (Figure 3). This technique allows to determine origin distances, fork speed and density on single DNA fibers.
The DNA replication checkpoint :
Role of Chk1 and Plk1 during unperturbed S phase
We and others have shown that the replication program is regulated by the ATR-dependent replication checkpoint pathway like in differentiated cells (Marheineke and Hyrien 2004). Blocked replication forks activated the ATR/Chk1 pathway which leads to the inhibition of late firing replication origins and a delay of S phase. We have further studied the role of the effector kinase Chk1 (Platel et al.,2015) and of Polo-like kinase 1 (Plk1) (Ciardo et al. 2020). We show that active Plk1 is recruited to chromatin before the activation of origins, positively regulates DNA synthesis and is highly abundant before MBT, when S phase is rapid due to a higher density of active origins. Recently, we revealed a new mechanism of how Plk1 promotes origin firing. Using different proteomic approaches we found that Plk1 interacts with a known negative regulator of the replication program, Rif1. Plk1 phosphorylates this factor on a critical residue necessary for its interaction with its subunit PP1, protein phosphatase 1. This leads to the dissociation of Rif1/PP1 from chromatin, the activation of the replicative helicase (MCM2-7 complex) and the activation of replication origins in genomic regions with high probability of origin firing or early replicating regions (Figure 4).
Figure 4 : A Example of combed DNA fibers (green) with replication eyes (red), from control (D mock) and Plk1 depleted (D Plk1) conditions, showing less replication tracks in the absence of Plk1. B Model of how Plk1 promotes origin activation via the inhibition of the Rif1/PP1 axis and the interaction with limiting initiation factors MTBP/Treslin/TopBP1.
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