Checkpoint Responses to DNA Double-Strand Breaks

Waterman, David P.; Haber, James E.; Smolka, Marcus B.

doi:10.1146/annurev-biochem-011520-104722

Cited by 125 publications

(116 citation statements)

References 240 publications

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“…Although we know a great deal about the evolutionarily related phosphoinositol-3-kinase like kinases ATM and ATR, including their structure and phosphorylation targets (29), the manner by which these kinases reach their targets have not been well characterized. In this paper, we used Bayesian model selection to distinguish how Mec1 and Tel1 are able to create extended regions of -H2AX after DNA damage.…”

Section: Discussionmentioning

confidence: 99%

“…Results similar to 1D sliding would also be predicted if chromatin near a DSB were actively extruded into a loop, a process different from the looping model in which the chromosome only undergoes passive looping due to thermal fluctuations. In vitro experiments have shown that chromatin can be actively pulled through the ring-shaped cohesin or condensin complexes, resulting in extruded chromatin loops that grow over time (29,30). Indeed, in yeast, cohesin and another SMC complex, Smc5,6 are recruited to sites of DSB damage (46)(47)(48).…”

Section: Discussionmentioning

confidence: 99%

“…where the "LM" subscript stands for the looping model. Substitute equation (11) We construct the model to be consistent with ChIP measurements of Tel1 and Mec1 at the break site, which show their levels increase linearly with time (15,29). These measurements could indicate two different dynamics.…”

Section: Derivation That Incorporates the Variability In The Timing Omentioning

confidence: 94%

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Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

Bronk

Kondev

et al. 2019

Preprint

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One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (-H2AX) around a DNA double-strand break (DSB). In the budding yeast S. cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1 ATR or Tel1 ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by ChIP-qPCR in order to uncover the mechanisms by which Mec1 ATR and Tel1 ATM propagate histone modifications across chromatin. With either kinase, -H2AX spreads as far as ~50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans -at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a 3D diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Derivation That Incorporates the Variability In The Timing Omentioning

confidence: 94%

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Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

Bronk

Kondev

et al. 2019

Preprint

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“…Cell cycle checkpoints arrest the self-replication process and increase the chances of survival when a cell encounters errors. 4,10,11 However, checkpoints show more complex behaviors than merely gating the cell cycle depending on the presence or absence of errors. After prolonged arrests, checkpoints such as the DNA damage checkpoint or the spindle assembly checkpoint (SAC) are overridden in the continued presence of errors, which is associated with genomic instability and aneuploidy in yeast and mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

“…Experiments support the existence of a balance between risk and speed qualitatively: Budding yeast cells with dysfunctional DNA damage (rad9∆) or spindle assembly checkpoints (mad2∆), which arrest very briefly 4 or not noticeably 10 , die at much higher rates in the presence of DNA damage or poor spindle-chromosome binding. 10 Multiple experimental challenges have to be overcome in order to resolve checkpoint strategies more quantitatively:…”

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confidence: 99%

Optimizing checkpoint strategies based on first principles predicts experimental DNA damage checkpoint override times

Sadeghi

Dervey

Gligorovski

et al. 2020

Preprint

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Why biological quality-control systems fail is often mysterious. Specifically, checkpoints such as the DNA damage checkpoint or the spindle assembly checkpoint are overriden after prolonged arrests allowing cells to continue dividing despite the continued presence of errors. Although critical for biological systems, checkpoint override is poorly understood quantitatively by experiment or theory. Override may represent a trade-off between risk and speed, a fundamental principle explaining biological phenomena. Here, we derive the first, general theory of optimal checkpoint strategies, balancing risk and opportunities for growth. We demonstrate that the mathematical problem of finding the optimal strategy maps onto the question of calculating the optimal absorbing boundary for a random walk, which we show can be solved efficiently recursively. The theory predicts the optimal override strategy without any free parameters based on two inputs, the statistics i) of error correction and ii) of survival. We apply the theory to the prominent example of the DNA damage checkpoint in budding yeast (Saccharomyces cerevisiae) experimentally. Using a novel fluorescent construct which allowed cells with DNA breaks to be isolated by flow cytometry, we quantified i) the probability distribution function of repair for a double-strand DNA break (DSB), including for the critically important, rare events deep in the tail of the distribution, as well as ii) the survival probability if the checkpoint was overridden. Based on these two measurements, the optimal checkpoint theory predicted remarkably accurately the DNA damage checkpoint override times as a function of DSB numbers, which we measured precisely at the single-cell level. Our multi-DSB results refine well-known bulk culture measurements and show that override is a more general phenomenon than previously thought. Further, we show for the first time that override is an advantageous strategy in cells with wild-type DNA repair genes. The universal nature of the balance between risk and self-replication opportunity is in principle relevant to many other systems, including other checkpoints, developmental decisions, or reprogramming of cancer cells, suggesting potential further applications of the theory.

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Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta‐Actin in Liver and Breast Cancer Cells

Tetikoğlu

Çelik-Uzuner

2023

Chemistry & Biodiversity

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Bee venom is a natural mixture and candidate anti‐cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β‐actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β‐actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA‐MB‐231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and β‐actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of β‐actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of β‐actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β‐actin.

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Checkpoint Responses to DNA Double-Strand Breaks

Cited by 125 publications

References 240 publications

Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

Yeast ATM and ATR use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break

Optimizing checkpoint strategies based on first principles predicts experimental DNA damage checkpoint override times

Bee Venom Induces the Interaction between Phosphorylated Histone Variant, H2AX, and the Intracellular Site of beta‐Actin in Liver and Breast Cancer Cells

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