The optimal strategy balancing risk and speed predicts DNA damage checkpoint override times

Sadeghi, Ahmad Reza; Dervey, Roxane; Gligorovski, Vojislav; Labagnara, Marco; Rahi, Sahand Jamal

doi:10.1038/s41567-022-01601-3

Cited by 10 publications

(8 citation statements)

References 54 publications

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“…In contrast to the permanent cell cycle arrest we observe in response to 2 DSBs, a recent study also using two HO-mediated persistent DSBs showed that cells in fact adapt (Sadeghi et al 2022). One difference between these two 2-DSB systems is the relative position of the DSBs, which might affect how SAC components become engaged, and thus might determine the extent of mitotic arrest.…”

Section: Resultscontrasting

confidence: 90%

“…Our previous results showed that when MAD2 was deleted, the length of cell cycle arrest was the same in strains with a single DSB, irrespective of the DSB’s distance to its centromere (Dotiwala et al 2010). We suggest that the strains used by (Sadeghi et al 2022) do not remain permanently arrested because one of the two DSBs in their strains is sufficiently far from its centromere to fully trigger SAC.…”

Section: Discussionmentioning

confidence: 93%

“…The fact that not all combinations of two DSBs produce permanent arrest (Sadeghi et al 2022) can be explained by the idea that the distance between the DSB site and its corresponding centromere is an important determinant of the extent of cell cycle arrest. We previously observed that a strain with a single DSB 200 kb away from the centromere had shorter cell cycle arrest compared to a strain with a DSB 86 kb away from the centromere (Dotiwala et al 2010).…”

Section: Depletion Of Mad1 Ormentioning

confidence: 99%

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Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint

Zhou

Waterman²,

Caban-Penix

et al. 2023

Preprint

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To prevent cell division in the presence of a DNA double-strand breaks (DSB), cell cycle progression is arrested by the DNA damage checkpoint (DDC) to allow more time for repair. In budding yeast, a single irreparable DSB arrests cells for about 12 h – 6 normal doubling times - after which cells adapt to the damage and resume the cell cycle. In contrast, 2 DSBs provoke permanent G2/M arrest. While activation of the DDC is well-understood, how it is maintained remains unclear. To address this question, key checkpoint proteins were inactivated by auxin-inducible degradation 4 h after damage induction. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2resulted in resumption of cell cycle, indicating that these checkpoint factors are required both to establish and to maintain DDC arrest. However, when Ddc2 is inactivated 15 h after inducing 2 DSBs, cells remain arrested. This continued arrest depends on the spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2. Although Bub2 acts with Bfa1 to regulate mitotic exit, inactivation of Bfa1 did not trigger checkpoint release. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC.

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Section: Resultscontrasting

confidence: 90%

Section: Discussionmentioning

confidence: 93%

Section: Depletion Of Mad1 Ormentioning

confidence: 99%

See 1 more Smart Citation

Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint

Zhou

Waterman²,

Caban-Penix

et al. 2023

Preprint

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“…Error correction plays a critical role in protein function and cell health by promoting accurate protein interactions and fixing damaged and incorrect genetic material. Previous work in such systems has focused on understanding the tradeoffs between energy, speed, and accuracy in error correction through kinetic modeling and experimental measurements of the timing of molecular events (e.g., enzyme dwell times, cell division times) as well as baseline error rates (4)(5)(6)(7). Measuring the dynamics of error correction in live cells is usually intractable due to the molecular scale and rapid speed of the processes involved.…”

Section: Introductionmentioning

confidence: 99%

Measuring and modeling the dynamics of mitotic error correction

Ha,

Dieterle,

Shen

et al. 2024

Preprint

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When functioning properly, the mitotic spindle segregates an equal number of chromosomes to daughter cells with high fidelity. Over the course of spindle assembly, many initially erroneous attachments between kinetochores and microtubules are fixed through a process called error correction. Despite the importance of chromosome segregation errors in cancer and other diseases, there is a lack of methods to characterize the dynamics of error correction and how it can go wrong. Here, we present an experimental method and analysis framework to quantify chromosome segregation error correction in human tissue culture cells with live cell confocal imaging, timed premature anaphase, and automated counting of kinetochores after cell division. We find that errors decrease exponentially over time during spindle assembly. A coarse-grained model, in which errors are corrected in a chromosome autonomous manner at a constant rate, can quantitatively explain both the measured error correction dynamics and the distribution of anaphase onset times. We further validated our model using perturbations that destabilized microtubules and changed the initial configuration of chromosomal attachments. Taken together, this work provides a quantitative framework for understanding the dynamics of mitotic error correction.

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“…Leakiness is a poorly characterized but crucial property since for many applications it is essential to be able to turn expression truly 'off'. Leakiness is particularly important when controlling genes that are toxic or cause changes to the genome such as Cas9 23 , Cre-loxP 11 , or Ho 24 endonuclease. However, for inducible systems, most of these properties have either not been assessed precisely, not in a manner that would allow their direct comparison, or have not been determined at all.…”

Section: Introductionmentioning

confidence: 99%

Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast

Gligorovski

Sadeghi

Rahi

2020

Preprint

Self Cite

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For quantitative systems biology, simultaneous readout of multiple cellular processes as well as precise, independent control over different genes’ activities are needed. In contrast to readout systems such as fluorescent proteins, control systems such as inducible transcription-factor-promoter systems have not been characterized systematically, impeding reliable modeling and precise system-level probing of biological systems.We built a comprehensive single-copy library of inducible promoters controlling fluorescent protein (yEVenus) expression in budding yeast, including GAL1pr, GALLpr, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr and the blue light-inducible systems EL222-LIP, EL222-GLIP. To track their properties under dynamic perturbations, we performed high-throughput time-lapse microscopy. The analysis of >100 000 cell images was made possible by the recently developed convolutional neural network YeaZ. We report key coarse-grained kinetic parameters, levels of noise, and effects on cellular growth. Our multidimensional benchmarking uncovers unexpected disadvantages of widely used tools, e.g., slow off kinetics of the doxycycline-induced tetOpr system, nomonotonic activity, or high variability of PHO5pr. Our data would guide the choice of acceptable compromises for applications. Evaluating the ARG3 promoter for potential use as a new inducible system, we discovered that it has an interesting OR gate function and that it turns on in the presence of methionine in synthetic complete medium. To demonstrate the ability to finely control genetic circuits, we tuned the time between cell cycle Start and mitotic entry in budding yeast experimentally, exogenously simulating near-wild-type timing.The data presented here ought to facilitate the choices of expression systems for quantitative experiments and applications in systems and synthetic biology and to serve as a reference to benchmark new inducible systems.

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The optimal strategy balancing risk and speed predicts DNA damage checkpoint override times

Cited by 10 publications

References 54 publications

Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint

Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint

Measuring and modeling the dynamics of mitotic error correction

Multidimensional single-cell benchmarking of inducible promoters for precise dynamic control in budding yeast

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