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A limited number of double-strand DNA breaks are sufficient to delay cell cycle progression.

Jeroen van den Berg, Anna G. Manjon, Karoline Kielbassa, Femke M. Feringa, Raimundo Freire, Rene H. Medema

Preprint posted on May 07, 2018 https://www.biorxiv.org/content/early/2018/05/07/316158

How many breaks do you need to stop a cell cycle? Utilizing CRISPR/Cas9, van den Berg et al. demonstrate that just one break is all it takes to slow a cell’s proliferation.

Selected by Leighton Daigh

Background

Cellular genomic integrity is constantly challenged by endogenous and exogenous threats, requiring persistent surveillance and repair. Cells possess numerous complex, intertwined pathways capable of resolving numerous types of DNA damage. Many of the cellular processes involved in the DNA damage response have been elucidated by classical studies using chemical mutagens and ionizing radiation to induce DNA breaks. While these methods proved effective for identifying the major components of the DNA damage response, the methods are also nonspecific and cause DNA damage in a broad and untargeted manner across the genome.

Inhibition of the cell-cycle following DNA damage is achieved through numerous pathways. The DNA damage-responsive kinases ATR and ATM induce a rapid inhibition of proliferation by activating a kinase cascade. Cyclin-dependent kinases (CDKs), which drive cellular proliferation, are quickly phosphorylated by downstream kinases ATR or ATM, resulting in an immediate inhibition of the cell cycle. The transcription factor p53 drives a more delayed inhibition of cellular proliferation following DNA damage through numerous mechanisms. One such mechanism is the transcriptional upregulation of p21, which halts proliferation by binding to and inhibiting CDK/Cyclin complexes.

Prior studies have attempted to achieve greater control in the genomic location of DNA breaks by using endonucleases targeting specific DNA sequences. However, these methods are limited by the availability of endonuclease target sites within the genome. Presently, it’s largely unknown how the precise number of DNA breaks and their timing at different cell-cycle phases affects cellular proliferation.

Key Findings

To address this knowledge gap, van den Berg et. al utilized the flexibility of CRISPR/Cas9-mediated cleavage at specified DNA sequences to determine how the precise number of DNA double-strand breaks affects the DNA damage response and cell-cycle progression. The authors first showed that CRISPR/Cas9 expression can be tightly controlled by combining Tet-On transcriptional control with destabilizing-domain post-translational control of the Cas9 nuclease, allowing tight temporal control over when in the cell cycle Cas9 is active. This was important, as it greatly decreased the background rate of Cas9-mediated DNA damage and improved sensitivity in the assay. A precise number of double-strand breaks could then be induced by choosing guide RNAs with a desired number of target sites in the genome. The authors validated the precision of this strategy by showing that the number of DNA damage foci observed by immunofluorescence closely correlates with the number of genomic sites targeted by a specified guide RNA (Figure 1).

Figure 1: Panels reproduced from Figure 2B of van den Berg et al., 2018 under a CC-BY-NC-ND 4.0 international license, representing DNA damage markers γH2AX and 53BP1. The number of genomic loci targeted with each guide RNA is indicated, ranging from 1 to 17.

 

The authors next evaluated the downstream consequences of inducing a single DNA break. They observed that inducing cleavage at one DNA locus was sufficient to activate the G1 or G2 checkpoint by inducing an ATM- and p53-driven DNA damage response. These checkpoints prevent cellular progression from G1- to S-phase or G2- to M-phase, respectively. Interestingly, damage that occurred in G1 appeared to result in more sustained inhibition of cellular proliferation, as compared to G2 damage. Cell-cycle arrest appeared to be temporary; the authors did not observe an increase in p21 or signs of cellular senescence that would indicate a more long-term inhibition of proliferation. These results make sense: cells regularly experience double-strand breaks and other DNA lesions. If an individual double-strand break caused long-term or permanent cell cycle arrest, there would be insufficient proliferative cells for the establishment and maintenance of tissue homeostasis. The authors conclude the preprint by demonstrating that inhibition of proliferation following a single DNA break is functionally important. If the DNA damage response is experimentally inhibited, cells undergo more irreversible DNA damage and lose their proliferative potential.

What I like about this preprint

This preprint nicely characterizes the use of CRISPR/Cas9 technology for time- and site-specific induction of DNA damage. The authors demonstrate the utility of this tool and validate the downstream effects of inducing an individual double-strand break. Importantly, this lays the groundwork for future studies to use CRISPR/Cas9 genomic targeting to determine if DNA double-strand breaks at specific genomic loci induce unique DNA damage responses. Moreover, the temporal control of the system enables further investigation of how a DNA break at a specified locus may induce differential DNA damage responses depending on cell states such as cell-cycle phase, cell differentiation, etc.

Questions for Authors

  • It’s possible that multiple rounds of cutting and repair occur at a targeted DNA locus before the gRNA binding sequence is destroyed by low fidelity repair. Is it possible to estimate exactly how many times the DNA is cut before the gRNA will no longer target Cas9 to the site?
  • The lack of p21 upregulation following Cas9 cleavage at a single site is intriguing. Is there a threshold for the number of DNA breaks that must occur before there is an upregulation of p21?
  • Could a Cas9 nickase be used to study the cellular response to single-strand breaks in an analogous manner?

Further Reading

  • Brinkman EK, Chen T, de Haas M, Holland HA, Akhtar W, van Steensel B (2018) Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks. Molecular Cell 70: 801-813.
  • Chao HX, Poovey CE, Privette AA, Grant GD, Chao HY, Cook JG, Purvis JE (2017) Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle. Cell Syst 5: 445–459.
  • Janssen A, Breuer GA, Brinkman EK, Van Der Meulen AI, Borden S V., Van Steense B, Bindra RS, Larocque JR, Karpen GH, Meulen AI Van Der, et al. (2016) A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and Euchromatin. Genes Dev 30: 1645–1657.
  • Purvis JE, Karhohs KW, Mock C, Batchelor E, Loewer A, Lahav G (2012) p53 Dynamics Control Cell Fate. Science 336: 1440–1444.

 

Posted on: 1st August 2018 , updated on: 4th September 2018

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