This method can be combined with depletion or inhibition of specific proteins, therefore uncovering the hierarchy of recruitment of different DNA repair factors to DSBs. Finally, approaches such as pulsed-field gel electrophoresis PGFE or single-cell gel electrophoresis also known as comet assay can also be combined with IR exposure to directly investigate DSB repair Figure 2.
This technique allows the separation of rather large DNA pieces by forcing them to pass through an agarose matrix in response to changing electric fields Schwartz and Cantor, ; Carle and Olson, Yeast chromosomes are small enough to be resolved in PFGE Schwartz and Cantor, ; Carle and Olson, ; Figure 2 , thus fragmentation due to DNA damage can be observed by the appearance of a smear of smaller bands Contopoulou et al.
The much larger mammalian chromosomes, on the contrary, remain on the wells during PFGE, and only smaller fragments caused by random DSBs will enter the gel Ager et al. The size distribution of the DNA portions is dependent on the number of breaks. A variation of this technique was developed in the Resnick laboratory using circular chromosomes in yeast or Epstein-Barr virus episomes in human cells Ma et al.
Another variation of this technique, the single-cell gel electrophoresis or comet assay, is a convenient way to estimate the number of DSBs created upon a given treatment with DNA damaging agents, such as IR, and to follow the kinetics of DNA repair in individual cells. Briefly, cells are treated with the DNA damage source, embedded in agarose to retain the nuclear structure, lysed and subjected to electrophoresis Olive et al.
DNA is attracted to the anode, but only broken fragments are small enough to abandon the nucleus Figure 2. After staining with a DNA dye, nuclei are observed with a fluorescent microscope and the displacement of DNA from the nucleus depends on the number of breaks per genome Olive et al.
By analyzing samples at different time points after DSB induction, the kinetics of repair can be estimated. Moreover, since radiation induces DSBs at unknown locations, and in a non-homogenous manner in the cell population, locus-specific analyses of DDR factor recruitment or chromatin modifications using chromatin immunoprecipitation ChIP studies, for instance, is not possible.
Thus, in all cases, appropriate controls must be used. For instance, ultraviolet A UVA radiation can be used to create hundreds of DSBs along the path of a laser beam line or spot through laser scanning microscopy Lukas et al.
Laser irradiation provides two main advantages. First, one can decide where to direct the laser beam in the nucleus, allowing to target specific subnuclear compartments, such as the nucleolus Kruhlak et al. Second, the concentration of hundreds of breaks along a laser track facilitates the observation of the recruitment of factors that either do not spread at all, or gradually increase over time, and for which foci are therefore difficult to see, especially at early time points.
As such, laser irradiation represents the most powerful tool to accurately determine the kinetics of DDR factors, providing a temporal resolution below 10 s.
Combined with the expression of fluorescently labeled proteins, laser microirradiation has provided unprecedented temporal resolution of the sequence of events following DNA damage Kochan et al.
The use of fluorescently-tagged histone proteins allowed the study of chromatin dynamics following damage with great resolution Burgess et al. Finally, this method has been useful to investigate the release of factors from DSBs and the post-translational modifications PTMs that drive such dynamics. In this case, the signal void created by the absence of the protein or by the removal of a specific PTM can be seen as a negative stripe or anti-stripe Chou et al. Moreover, it is neither amenable for molecular characterization of the repair outcome at the sequence level, nor for ChIP, which limits the spatial resolution that can be achieved.
In contrast to radiation treatments, that require specialized and expensive equipment, chemical-induction of DSBs is cheap and easy to implement in any laboratory and can be coupled with almost any experimental protocol. Usually, cells are treated with a defined concentration of a chemical agent for a fixed amount of time.
It is important to distinguish between acute from minutes to a few hours versus chronic for days treatments, as the responses will vary enormously. Many types of chemical agents can indirectly cause DNA breaks. SSBs caused by camptothecin, a common inhibitor of topoisomerase I, can, in turn, be converted to DSBs during replication. Replication inhibitors, such as HU or aphidicolin, and crosslinker agents, like cisplatin or mitomycin C, can also cause one ended DSBs due to fork collapse Saintigny et al.
Additionally, several chemical agents imitate the effect of ionizing radiation and break the DNA directly Figure 2. These radiomimetic drugs include bleomycin, phleomycin or neocarzinostatin Sleigh, ; Edo and Koide, ; Chen and Stubbe, For instance, DSBs created by radiomimetic drugs show a bias toward specific sequences Murray and Martin, ; Burden et al. Moreover, topoisomerase II poisons such as etoposide preferentially induce lesions at CTCF binding loci located close or within transcriptionally active units Canela et al.
Topoisomerase I and replication inhibitors induce DSBs specifically during S phase or the following mitosis Saintigny et al. The analysis of DSBs induced by chemical agents can be performed by the same approaches described for irradiation-induced breaks Figure 2.
These techniques are well suited to investigate DSBs that occur non-randomly across the genome such as those induced by topoisomerase II poisons for instance. Of importance they not only provide an information about DSBs positions on the genome, but they are also quantitative, hence providing an estimate of break frequency in the cell population Aymard et al.
Different labs have sought to develop tools for the site-specific induction of DNA breaks making use of restriction enzymes targeting integrated exogenous cleavage sites, otherwise absent from the genome. Such tools overcome the ambiguity of DNA lesions introduced by previous methods and allow the inspection of protein recruitment during the DDR to a site-specific DSB and the assessment of chromatin remodeling events with nucleosome resolution.
Moreover, they can be combined with strategies to control the timing of DSB induction, for instance by controlling the nuclear translocation of the restriction enzyme, affording a valuable strategy to measure kinetic parameters of the DDR in live cells Berkovich et al. The first reporter system, employing a site-specific DSB at a reporter transgene integrated into the genome of mammalian cells was developed in the mids.
Following this seminal work, a large number of labs further developed similar strategies based on I- Sce I cut of a transgenic locus to investigate various aspect of the DDR, including repair pathway preferences and efficiency Gunn and Stark, ; Gelot et al.
For example, Soutoglou et al. For that, stable cell lines derived from mouse embryonic fibroblasts NIH3T3 were generated containing a single I- Sce I restriction site flanked by arrays of lac-repressor binding sites and tetracycline-response elements L-I- Sce I-T array Figure 3.
Expression and binding of fluorescently-tagged lac and tetracycline-repressors to these arrays enabled the simultaneous detection of both DNA ends. Figure 3. Schematic overview of methods to induce annotated DNA breaks at transgenic loci inserted in the genome. Examples of reporter genes that allow the direct inspection of DSB repair pathways and transcription and chromatin dynamics are represented.
Experimental approaches that can be coupled with the methods to induce DSBs at transgenic loci are shown.
Additional systems were further developed to generate multiple DSBs on a specific transgene, thus rendering the DNA repair easier to visualize. The Greenberg lab developed a noteworthy single-cell assay specifically designed to simultaneously analyze both the DSB repair and its effects on local transcription.
The experimental procedure was based on the introduction of multiple nuclease-induced DSBs upstream the promoter of an inducible transgene, modified to enable the visualization of transcriptional and translational events Shanbhag et al.
The reporter system, integrated in the genome of a human osteosarcoma U2OS cell line, is visualized upon binding of the mCherry-fluorescently-tagged lac-repressor protein mCherry-LacI to a lac-operator array. Nascent transcription is visualized by the accumulation of fluorescent MS2-binding proteins at the transcription site, upon binding to nascent MS2 stem-loop structures present at the reporter gene RNA Shanbhag et al.
Of note, this approach leads to persistent and extensive DSB induction and the time of damage induction is dependent on the expression of mCherry-LacI- Fok I.
A similar system to study transcription in proximity to DSBs was engineered by Ui et al. Upon tamoxifen treatment, the mCherry-tTA-ER fusion proteins translocate into the nucleus and localize at transcription sites TRE sites , to induce transcription activation, detected by the accumulation of fluorescently tagged-MS2 protein Rafalska-Metcalf et al.
Using this experimental system the authors reported a DSB-induced transcriptional repression mechanism involving the transcription elongation factor ENL Ui et al. A single I- Sce I restriction site was inserted in either the promoter-proximal region or within an internal exon of a reporter gene.
In addition to live-cell microscopy imaging, these reporters may be combined with ChIP-qPCR, providing a valuable tool to directly inspect the recruitment of DNA repair factors to a DSB, to assess histone modifications or measure nucleosome occupancy at broken ends. They can be combined with additional reporters to investigate with great detail the functional links between the DDR and transcriptional activity, chromatin modification and spatial organization, or DNA replication, for instance.
Consequently, they cannot provide the same temporal resolution achieved using microirradiation, where DSB induction is immediate and highly synchronized. Moreover, the transgenic nature of the analyzed loci calls for caution, especially when repeat-rich transgenes are used creating either multiple clustered DSBs or a single DSB but in a highly repeated transgenic locus, which may display a peculiar chromatin structure.
Finally, the accurate repair of endonuclease-created breaks reconstitutes the target site, therefore being re-cleavable until the target site has been mutated. Hence, most of the outputs measured in these experimental contexts address mutagenic repair, leaving faithful repair out of reach.
In order to bypass the need for introducing a transgene and to avoid potential, non-generalizable, side effects of transgenic loci on the repair process e.
Figure 4. Given that these DSBs are induced at annotated positions and in a homogeneous manner in the cell population, one can use ChIP to investigate protein recruitment at the site of damage.
This can also be coupled to high throughput sequencing analyses to investigate simultaneously repair events at multiples breaks ChIP-seq.
The Kastan lab developed a system that uses the eukaryotic homing endonuclease I- Ppo I, which has a recognition sequence of 15bp, to form site-specific DSBs within endogenous target sites of the human genome Berkovich et al. The addition of 4-hydroxytamoxifen 4-OHT promotes rapid nuclear localization of ER-I- Ppo I and the subsequent time-dependent cleavage of the endogenous sites. I- Ppo I has been further applied to interrogate DSB repair mechanisms in other organisms, such as fission yeast Sunder et al.
Of interest, in the latter, both temporal and spatial regulation of I- Ppo I activity was achieved by using a GFP-I- Ppo I endonuclease fused to an ER domain for tamoxifen-dependent temporal induction and whose tissue-specific expression was dependent on Cre recombinase. The results obtained using this in vivo model system showed transient, and DDR-dependent, decrease in gene expression of break-bearing - but not more distant - genes, further reversed upon DSB repair Kim et al.
Another DSB-inducible tool developed to create multiple endogenous, sequence-specific breaks, makes use of the Asi SI - 8bp cutter - restriction enzyme. The Legube lab, fused Asi SI to a modified ER ligand-binding domain, which controls nuclear localization of Asi SI—ER fusion protein, and to an auxin-inducible degron enabling controlled ubiquitination and degradation of the enzyme Iacovoni et al. This system is then amenable to compare DNA repair at various genomic positions. Importantly, while Asi SI is not able to damage heterochromatin, likely due to both the DNA methylation status and decreased accessibility of compacted chromatin Iacovoni et al.
Consequently it has been extensively used to provide high-resolution maps of repair proteins and chromatin changes Iacovoni et al. Hence while being powerful to analyze the spatial distribution of repair protein and chromatin changes around DSBs, they preclude a fine temporal resolution of these events.
Moreover, the position of the DSBs is dictated by the target site of the chosen enzyme, which can represent a limitation to the number of different loci analyzed. A number of specific tools have more recently allowed to induce DSBs at chosen endogenous genomic loci.
To introduce DSBs at specific loci of interest, it is possible to fuse the Fok I endonuclease to a protein able to specifically target a particular locus. Zinc finger nucleases ZNF are chimeric proteins comprised of both a zinc finger domain designed to recognize a specific locus and the Fok I nuclease. TALE proteins were discovered as composed of a succession of 34aa monomers, each displaying the ability to recognize one nucleotide.
Indeed, recent studies indicated that Cas9-induced DSBs display highly mutagenic repair with nearly no accurate repair events Brinkman et al. As for the other endonuclease-mediated DSB induction systems, they are amenable to both imaging and molecular high throughput sequencing-based technologies such as ChIP-seq. Moreover, the fact that Cas9-induced DSBs may be particularly refractory to repair, and hence biased in terms of repair pathway choice, call for caution when using these systems.
Coating of telomeres with shelterin factors including telomeric repeat-binding factor 2 TRF2 , prevents fusions of linear chromosome ends and suppresses local DNA damage responses de Lange, Dysfunctional telomeres induce cellular responses that are highly similar to the ones elicited by DSBs, such as DDR activation and cellular senescence Fumagalli et al.
Dysfunctional telomeres can be generated through telomere uncapping and other forms of telomere damage, which may be specifically induced to activate the DDR in cycling cells. A plethora of methods - ranging from the visualization of DNA repair factors foci using immunofluorescence to the biochemical characterization of DDR complexes assembled at dysfunctional telomeres using ChIP - can be coupled to the TRF2 inactivation to investigate the molecular details of different aspects of the DDR.
Importantly, dysfunctional telomeres have been instrumental to discover the function of various proteins in DSB repair [e. The number of dysfunctional telomeres may vary considerably between cells and this heterogeneity may raise issues related with cells viability. Our capacity to create DSBs in a programed manner and in such a way that is compatible with a set of diverse methodologies to investigate the events that follow DNA damage, has led to our current deep understanding of the DDR.
The induction of DSBs at random locations using different sources of radiation or genotoxic compounds, provides the easiest approach to analyze the recruitment kinetics of proteins to sites of DNA damage and is a powerful strategy to temporally resolve the sequence of DNA repair events. Yet all the tools described here display significant drawbacks.
A major challenge is now to refine these DSB-inducible systems and the subsequent methodologies to analyze repair in order to overcome these limitations. All authors contributed to discussing the review contents and to writing the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ager, D. DNA double-strand breaks measurement of radiation-induced by pulsed-field gel electrophoresis. Cell Rep. Google Scholar. Aleksandrov, R. Protein dynamics in complex DNA lesions. Cell 69, — Aymard, F. Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes.
Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Ayrapetov, M. DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin. Bartke, T. A chromatin-based signalling mechanism directs the switch from mutagenic to error-free repair of DNA double strand breaks. Berkovich, E. Cell Biol. Biernacka, A. Blackford, A. Cell 66, — Bouquet, F. Cell Cycle 5, — Bouwman, B.
Genes 9:E Brinkman, E. Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks. Cell 70, — Britton, S. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. Burden, D. Topoisomerase II-etoposide interactions direct the formation of drug- induced enzyme-DNA cleavage complexes.
Burger, K. Nucleic Acids Res. Burgess, R. Activation of DNA damage response signaling by condensed chromatin. Burma, S. Canela, A. Thousands of DNA lesions can appear per cell per generation in an aerobic bacterial culture, and hundreds of thousands can appear in a single mammalian cell in a day Hoeijmakers, Most cells have DNA repair systems to enforce genome stability and, in higher eukaryotes, to prevent cancer.
However, these systems can break down Putnam et al. The widespread mutations and rearrangements of chromosomes that are found in tumour cells prevail in what can only be described as genomic chaos Carter et al. Documenting these various genomic insults and their consequences represents a major challenge for medicine, and also for disciplines such as evolutionary biology and cell biology. Now, in eLife , Susan Rosenberg of Baylor College of Medicine and colleagues—including Chandan Shee as first author—have provided a promising new tool for the study of one such insult: the double strand break Shee et al.
Double strand breaks are considered the most dangerous of all the DNA lesions. If left unrepaired, the resulting chromosome discontinuity often results in death. There are two main ways to repair a double strand break.
Recombinational DNA repair is accurate but it relies on the presence of an unbroken homologous chromosome. Non-homologous DNA end-joining, on the other hand, repairs the break, but usually at the expense of adding or deleting genetic information Chapman et al. Dangerous as they are, double strand breaks are sometimes deliberately introduced into a chromosome.
In yeast, directed double strand breaks are a prelude to an intrachromosomal exchange of genetic information that produces a mating type switch Haber, Double strand breaks are also central to genetic elements called transposons, and in genomic rearrangements that are integral to the immune system.
Accurate real-time detection of double strand breaks in a cellular genome is thus of great interest in the continuing effort to understand genome maintenance and function. A variety of techniques have been developed to detect and quantify double strand breaks, but they all have one or more deficits in terms of utility, efficiency, sensitivity or specificity.
Shee, Rosenberg and colleagues—including co-workers from the University of Texas, the MD Anderson Cancer Center and the University of Minnesota—now report a new approach, based on a protein called Gam, that offers some substantial advantages over existing approaches Shee et al.
Gam is encoded by the bacteriophage Mu: this is basically a hybrid of a bacterial virus and a transposon, and it makes a living by moving efficiently within and between bacterial genomes Baker, ; di Fagagna et al. When an integrated genomic copy of Mu replicates and transposes, the Gam protein protects the free ends of the Mu chromosome as they are transiently exposed. Gam is related to two eukaryotic proteins, Ku70 and Ku80, that are involved in non-homologous DNA end-joining.
Whereas the Ku proteins bind to double strand ends, they also interact with an array of other eukaryotic proteins and DNA structures, rendering them less useful for development of a general reagent that binds to double strand breaks.
DNA Repair Amsterdam 2 , — Pannunzio, N. RAD59 is required for efficient repair of simultaneous double-strand breaks resulting in translocations in Saccharomyces cerevisiae.
DNA Repair Amsterdam 7 , — Rothstein, R. Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae. Molecular and Cellular Biology 7 , — Schwartz, D. New techniques for purifying large DNAs and studying their properties and packaging.
Shrivastav, M. Regulation of DNA double-strand break repair pathway choice. Cell Research 18 , — Singh, R. Radiation Research , — Strout, M. The partial tandem duplication of ALL1 MLL is consistently generated by Alu-mediated homologous recombination in acute myeloid leukemia.
Proceedings of the National Academy of Sciences 95 , — West, S. Molecular views of recombination proteins and their control. Nature Reviews Molecular Cell Biology 4 , — Westmoreland, J. RAD50 is required for efficient initiation of resection and recombinational repair at random, gamma-induced double-strand break ends. PLoS Genetics 5 , e Eukaryotes and Cell Cycle. Cell Differentiation and Tissue.
Cell Division and Cancer. Cytokinesis Mechanisms in Yeast. Recovering a Stalled Replication Fork. Aging and Cell Division. Germ Cells and Epigenetics. Cristina Negritto, Ph. Citation: Negritto, M. Nature Education 3 9 Double-strand breaks in DNA can be lethal to a cell. How do cells fix them? Aa Aa Aa. Figure 1. DSBs can be repaired using several different mechanisms. When cells are treated with a DNA damaging agent that causes DSBs, the original chromosomal bands get broken into smaller fragments.
This process translates into an increase in intensity of smears between chromosomal bands, a smear toward the bottom of the gel, or both. Because bigger chromosomes are more susceptible to breakage, slowly migrating bands bigger chromosomes clearly visible in the control samples are the first ones to disappear or to appear as less intense in the treated samples Westmoreland et al.
Another approach to introducing DSBs in yeast cells is by using phenolic compounds. Figure 3 shows an example of this type of PFGE data. Compare lanes 1 and 3 at the top of the figure, where the largest chromosomal bands would appear, and note that damaged DNA is visible in one of the phenolic-treated samples. The effect of the phenolic treatment is not as drastic as that observed when treating cells with IR, as mentioned above. Figure 3: Induction of genome-wide DSBs.
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