CE12 - Génétique, génomique et ARN

CyclinA-Cdk1/2 Actors, from temporal control of DNA Replication to checkpoint adaptation and Mitotic Entry under stress – CARE-ME

Submission summary

Genome maintenance, cell identity and fate rely on the ordered execution of faithful DNA replication and accurate chromosome segregation across generations. Duplication of large genomes raises specific problems due to inherent limitations of several components of the replication machinery and relies on the sequential activation of replication origins following a precise spatio-temporal order. Although overall Cdk (Cyclin-dependent kinase) activity is required for the execution of the initiation program, the respective roles of the different, notably CyclinA2-Cdk2 and CyclinA2-Cdk1, complexes that assemble sequentially along S phase as well as the underlying regulatory mechanisms remain poorly understood. Proliferating cells routinely faces various threats challenging DNA replication and leading to fork slowing or stalling, situations refereed to replication stress (RS). Previous work established that cells respond by increasing the density of initiation events to complete DNA replication of fired domains, while activation of DNA replication checkpoint (DRC) inhibits S-phase Cdks, blocking initiation of yet unfired domains and preventing cell cycle progression. However, these findings do not take into account that RS stringency may finely tunes the level of CyclinA2-Cdk2 and/or -Cdk1 activities and consequently the regulation of extra initiation events.
- We recently found that, under some conditions, Cdk1 inhibition triggers extra-initiation events in expressed and late-replicating large genes, including those hosting common fragile sites (CSFs). Therefore, in addition to delay mitosis, Cdk1 inhibition accelerates replication of some hard-to replicate-regions, suppressing their instability.
- We developed a specific FRET (Förster Resonance Energy Transfer)-based Chk1 sensor to record DRC signaling over time in live single cell assays. We found that Chk1 activity is tightly modulated during normal S phase and upon incremental RS. Additionally, our approach revealed heterogeneous cell-to-cell Chk1 activity profiles along S to M progression during RS recovery that supports temporal activation of repair mechanisms up to mitosis onset.
Here, building on genetically engineered cells to specifically manipulate individual CyclinA2-Cdk complexes and a panel of FRET-based sensors, notably of Chk1 to record DRC signaling, we propose: (i) to decipher how CyclinA2-Cdk1 and CyclinA2-Cdk2 individually activate or repress both normal and RS-dependent replication origins to maintain genome stability. (ii) to characterize the repair mechanisms at work to perverse genome integrity upon RS recovery, and (iii) to tackle how under-replicated regions fail to prevent mitosis in DRC-proficient cells under RS, a main unanswered biological issue.

Project coordination

Olivier GAVET (Stabilité génétique et oncogenèse)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


Stabilité génétique et oncogenèse
Stabilité génétique et oncogenèse

Help of the ANR 483,300 euros
Beginning and duration of the scientific project: February 2021 - 48 Months

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