Quantifying DNA double-strand breaks induced by site ... - Nature

protocol

Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification Catherine Chailleux1,2, François Aymard1,2, Pierre Caron1,2, Virginie Daburon1,2, Céline Courilleau1,2, Yvan Canitrot1,2, Gaëlle Legube1–3 & Didier Trouche1–3 1Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. 2Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France. 3These authors contributed equally to this work. Correspondence should be addressed to

D.T. ([email protected]).

© 2014 Nature America, Inc. All rights reserved.

Published online 6 February 2014; doi:10.1038/nprot.2014.031

Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.

INTRODUCTION DNA DSBs are the most deleterious form of DNA damage, and they can lead to mutations or chromosomal rearrangements. Proteins involved in DNA damage management or repair are often mis-expressed in human cancers, and mutations in these proteins are the molecular causes of cancer-prone syndromes (such as ataxia telangiectasia, xeroderma pigmentosum, Nijmegen breakage syndrome and so on)1,2. In addition, many current anticancer treatments rely on the use of DSB-generating compounds or radiations. As a consequence, the molecular mechanisms involved in DSB management often dictate the efficiency of such treatments, and they are currently the subject of intense research, both for deciphering basic mechanisms and for clinical anticancer research1,2. Historically, random DSBs were induced by chemical or physical sources, and their appearance and repair were followed by analytical techniques such as comet assays3. These studies proved to be very useful for the analysis of the efficiency of global DNA DSB repair and the contribution of specific proteins or drugs to this efficiency. However, the major limitation of such treatments is that DNA DSBs are induced in a random manner in the genome, and only information on global parameters of DSB repair can be obtained by such methods. Recently, cell lines or organisms4,5 have been constructed in which DSBs can be induced by tightly regulated restriction enzymes, leading to the generation of sequence-specific DSBs. These recombinant restriction enzymes can be produced through transient transfection of an expression vector, or they can be expressed in an inactive form through a transgene, being activated upon the addition of an exogenous ligand. Such systems were originally developed to study the involvement of proteins such as BRCA1 in DSB repair by homologous recombination (HR)6. Since then, they have been widely used to investigate the recruitment

of repair factors or chromatin modifications around DSBs by techniques from the chromatin field, such as conventional chromatin immunoprecipitation (ChIP, see for example, refs. 7–11), ChIP-chip12,13 or ChIP-seq14. These restriction enzyme–based systems have also allowed the fate of DSBs to be analyzed by imaging techniques15, leading to insights into their subcellular localization. Finally, reporter systems based on transgenes containing DNA sequences specifically digested by the endonuclease I-SceI (which does not cut the human genome) have been developed to rapidly and precisely assess the efficiency of the various DSB-repair pathways, such as HR, nonhomologous end-joining (NHEJ) or alternative NHEJ16–19. Such reporter systems are now largely used by the scientific community to assess the contribution of a given protein to these DNA-repair pathways (see for example, refs. 16,20–25). The use of enzyme-induced breaks thus allows unique and highly valuable information to be obtained. The major drawback of these restriction enzyme–based systems compared with conventional chemical or physical DSB-inducing treatments is that the digestion efficiency of the restriction enzyme can be affected by cell treatments (depletion of the proteins by siRNA, treatment with inhibitors and so on; Supplementary Fig. 1). Indeed, these treatments can, for example, affect the expression of the restriction enzyme and/or the chromatin structure around the restriction enzyme sites. Thus, the quantity of breaks mediated by the restriction enzyme and the number of breaks present at a given time must be measured with maximum accuracy to adequately interpret these experiments. Ligation-mediated purification and its applications Here we present a method that we have named ligationmediated purification, which is based on purification of enzymatically cleaved DNA, followed by its quantification (see Fig. 1 nature protocols | VOL.9 NO.3 | 2014 | 517

protocol Genomic DNA with sequence-specific DSB

Step 1: in cellulo enzyme cutting

Step 2: purification of genomic DNA

Steps 3–5: ligation with the cohesive biotinylated oligonucleotide

Step 6: DNA fragmentation by restriction enzyme A

Enzyme A


Steps 14–16: precipitation with streptavidin-coated beads

Step 17: elution from beads with restriction enzyme B Enzyme B

Enzyme B

Step 21: quantification of specific DNA sequences Option A: real-time PCR quantification

Option B: genome-wide analysis

Figure 1 | General scheme of the experimental procedure with the main steps highlighted.

for a general scheme of the technique). Briefly, sequence-specific DNA breaks are introduced in living cells through the controlled activity of a site-specific endonuclease. A biotinylated oligonucleo tide with a cohesive end compatible with the enzyme-induced DSB (Fig. 2) is ligated to enzymatically cleaved genomic DNA, followed by purification (pull-down) of ligated DNA on streptavidin beads. Pulled-down DNA is then analyzed by quantitative techniques such as real-time PCR or by genome-wide strategies (such as microarrays or high-throughput sequencing). Given its accuracy and linearity (Fig. 3), as well as reproducibility (Fig. 3d), this technique can be used to compare the levels of restriction enzyme–mediated breaks between various genomic sites in the same cell population. As an example, we used it to better-characterize the efficiency of AsiSI-mediated DSB induction that occurs in the AsiSI-estrogen receptor (ER)

Figure 2 | Design of biotinylated oligonucleotides. (a) General design of the biotinylated oligonucleotides. The end cohesive with the enzyme used to generate DSBs is in red, and the site for the enzyme used to elute the biotin-labeled DNA (enzyme B) is in blue. The biotin label is indicated by a green diamond. (b) Examples of the I-SceI and AsiSI oligonucleotides used in our studies. Numbers inside blue circles in both panels correspond to the items listed in a. 518 | VOL.9 NO.3 | 2014 | nature protocols

a

inducible cell line13. Nuclear relocalization of the AsiSI restriction enzyme by hydroxytamoxifen treatment should lead to the generation of DSBs at more than 1,000 AsiSI annotated sites on the human genome. However, by using the cleavage assay followed by hybridization on microarrays covering chromosomes 1 and 6, we found that only ~20% of annotated sites are efficiently cleaved in vivo. In addition, the correlation of these data with the profile of phosphorylated H2AX (γH2AX, a marker for DSBs) revealed that AsiSI-mediated DSBs are always associated with γH2AX13, irrespective of their genomic location. More importantly, this technique can also be used to assess the effect of a treatment (such as protein depletion by siRNA) on the induction of cleavage at a given site. For example, we recently observed a striking increase in γH2AX levels around two AsiSIinduced breaks after treatment with a DNA protein kinase (DNAPK) inhibitor (Supplementary Fig. 1). However, in such conditions, ligation-mediated purification revealed a parallel strong increase in the levels of DSBs present at these sites (Supplementary Fig. 1). Controlling the extent of cleavage is thus absolutely essential to avoid misinterpretation of results. In contrast, in another study, we found that depletion of cohesin by siRNA affected γH2AX spreading, without changing the quantity of breaks induced, thus allowing us to conclude that cohesin controls γH2AX distribution12. More recently, we adapted this technique to DNA-repair reporter systems relying on a transgene containing one or two I-SceI sites. We were able to show that the depletion of p400 (an ATPase acting as a chromatin remodeler) affects HR efficiency measured with a reporter vector, without affecting the amount of I-SceI–mediated breaks26. We also obtained measurements of I-SceI–mediated DNA cleavage on other reporter systems, including an NHEJ reporter system (Fig. 3a,b). It is, however, important to understand that data generated by ligation-mediated purification is a snapshot of the presence of enzyme-mediated breaks at a given time; this value depends on the amount of breaks generated by the enzyme (accessibility to the chromatin, efficiency of the enzyme) and also on the efficiency of DNA repair. Thus, differences in the levels of enzyme-mediated breaks between various conditions or at various genomic sites cannot be easily used to raise conclusions about repair defects. Rather, it provides an essential control for correctly analyzing endonuclease-based experiments such as ChIP (Supplementary Fig. 1 and refs. 12,13) or I-SceI–based HR26 and NHEJ assays. Overview of the procedure DNA cleavage is induced in cultured cells by the expression of an active restriction enzyme, and then the cleaved genomic DNA is purified. A biotinylated oligonucleotide is then ligated to the purified DNA. The oligonucleotide is designed to have a cohesive

Biotinylated double-strand oligonucleotide

1. Biotinylated end 2. Restriction enzyme site (enzyme B in Fig. 1) for elution 3. Single-strand end cohesive with cleaved genomic DNA

b

I-SceI-biotinylated oligonucleotide

AsiSI-biotinylated oligonucleotide

protocol

0.5 0.4 0.3 0.2 0.1

3 2 1 0

–I -S ce +I I -S N ce o I lig as e



–I -S c +I eI -S ce I

0


4

I-SceI site

Internal control (1/20)

Control genomic sequence

I-SceI site

c

d

40

100 30 y = 1.026 × –0.2

20

10

0

0

10

20

30

40

% of in vitro– cleaved relative to uncleaved genomic DNA

Amount of AsiSI-cohesive breaks (%)

0.6

5

Amount of I-SceI–cohesive breaks (%)

b

0.7

Amount of I-SceI–cohesive breaks (%)

Pulled-down DNA (% of input)

a

80 60 40 20

0 OH-Tam

– +

– +

DSB-1 DSB-2

Figure 3 | Typical experiments analyzed by qPCR. (a) GC92 cells, which contain a transgene with two I-SceI sites designed to assess NHEJ17, were transfected by an I-SceI expression vector (+I-SceI) or by the control empty vector (–I-SceI). Forty-eight hours later, genomic DNA was prepared and subjected to ligation-mediated purification with the I-SceI oligonucleotide described in Figure 2b. A –ligase sample was performed by using the genomic DNA prepared from the +I-SceI samples. The amounts of the indicated sequences (at the vicinity of a transgenic I-SceI site (I-SceI site), on the internal control and on a control genomic sequence) were quantified by qPCR. Percentages relative to input are shown. Error bars represent s.d. of the qPCR measurements done on triplicates. For clarity, the values for the internal control were divided by 20. Note that the amount of I-SceI sequence in the +I-SceI was higher than the three controls (‘–I-SceI’, ‘no ligase’ or amplification of the ‘control genomic sequence’). (b) Quantification of the extent of cleavage at the I-SceI site in the experiment shown in a. The percentage relative to inputs for the I-SceI site was divided by the percentage relative to inputs for the internal control for each sample. Considering that cleavage of the internal control is 100%, absolute percentages of cleavage are shown. Error bars represent s.d. of the qPCR measurements. Note that ~4% of I-SceI sites are cleaved after transfection of the I-SceI expression in this experiment. (c) Genomic DNA purified from GC92 cells was prepared and digested in vitro by using the I-SceI enzyme (digestion was total, as checked by qPCR using primers present on both sides of the break; data not shown). Cleaved genomic DNA was mixed with uncleaved genomic DNA at the indicated percentages and subjected to ligation-mediated purification followed by qPCR as in a. Absolute percentages of cleavage are plotted. A representative experiment out of four is shown. Note the accuracy (a 30% mix of cleaved DNA relative to uncleaved DNA is measured as 31% of cleavage) and the linearity (indicated by the 1.02 coefficient of the regression) of the method. (d) U2OS cells expressing ER-AsiSI were subjected to ligation-mediated purification without or with DNA breaks induction (induced by a 4-h treatment by OH-Tam). The amount of sequences near two AsiSI sites and on the internal control was measured in the samples and in the inputs by real-time PCR. Absolute percentages of cleavage were measured around two AsiSI sites by qPCR. The mean and s.d. from six biological replicates are shown.

end complementary to the cohesive end generated by the restriction enzyme used for DSB induction (for AsiSI or I-SceI, see the sequences of the oligonucleotides we routinely use in Fig. 2b): thus, only DNA cut by the enzyme will be ligated to the oligo nucleotide. This ligation step specifically tags the breaks induced by the enzyme with biotin for later purification. Next, the tagged genomic DNA is fractionated to obtain DNA fragments of ~5,000 bp; it is necessary to limit the size of the biotinylated DNA so that only sequences located close to the breaks are specifically tagged. Although other methods, such as sonication, may be used, we use restriction enzyme digestion (restriction enzyme A in Fig. 1, in our case either EcoRI or ScaI), as it allows a precise limit of biotinylated DNA fragments to be purified, which is useful for genome-wide analysis of pull-down DNA (see for example results from Fig. 4). Any enzyme can be chosen, provided that it does not cut between the DSB of interest (either in genomic DNA or in the internal control) and the primers used for detection by qPCR. The biotinylated oligonucleotide (ligated to genomic DNA fragments) is then pulled down with streptavidin beads. The strength of the biotin-streptavidin interaction allows very stringent washes, and thus it decreases background caused by binding of genomic DNA to beads. Ligated genomic DNA is eluted by digesting with a restriction enzyme whose site is included in the biotinylated oligonucleotide (restriction enzyme B in Fig. 1, in our case HindIII). This step is required because of the very strong affinity of the biotin-streptavidin interaction, which cannot be efficiently disrupted.

Next, eluted DNA is precipitated, and the presence of the sequence near the DSB of interest is determined by qPCR. Given that the goal is precise quantification of the amount of breaks induced at a specific site, real-time PCR is the method of choice for analyzing the results. It allows the calculation, as for ChIP experiments, of the ratio between pulled-down and input DNA (the input genomic DNA also being analyzed by qPCR; see an example in Fig. 3a). Purified DNA samples can also be analyzed by hybridization on tiling arrays, which allows the efficiency of DSB induction to be directly assessed at all sites simultaneously. This can be particularly useful when an enzyme that generates multiple DSBs (such as AsiSI) is used to induce breaks in vivo. In this case, DNA is amplified and labeled (for example, according to the procedure described in the Affymetrix chromatin immunoprecipitation assay protocol, available at http://www. affymetrix.com/estore/browse/products.jsp?productId=131463 &categoryId=35848&productName=GeneChip-Human-Tiling2.0R-Array-Set1_3) before hybridization on microarrays. The array data obtained with the DNA recovered after streptavidin pull-down is normalized against the data obtained by using the input sample. For each DSB annotated position (for example, AsiSI sites), the signal encompassed between the two flanking EcoRI sites is calculated (provided that EcoRI was used as enzyme A; Fig. 4). Although this approach cannot give an absolute percentage of cleavage efficiency like qPCR analysis, it allows DSB induction levels to be compared between sites across the genome. Similar analysis can probably be performed by highthroughput sequencing. nature protocols | VOL.9 NO.3 | 2014 | 519

protocol


Limitations The main limitation of ligation-mediated purification is that it might not detect DNA breaks that have been processed by the repair machinery. As a result, our technique may underestimate the percentage of broken DNA. Given that when DNA breaks are induced by using restriction enzymes the broken ends are cohesive, repair by NHEJ can probably occur without any processing of DNA ends. However, HR, the alternative major DSB-repair pathway, requires processing of DNA ends: it begins with DNA resection to create single-stranded DNA, which can then invade the unbroken sister chromatid to form Holliday junctions. Repair is completed after the resolution of Holliday junctions. These HR-dependent intermediates might not be efficiently detected by our methodology. To test whether this limitation could be a major concern, we compared the outcome of our technique with conditions in which the extent of resection is modulated by using siRNAs against CtIP, which is a protein required for this process27,28. We found that the inhibition of CtIP expression does not change the amount of breaks measured by our assay at two AsiSI sites in U2OS cells at early time points (4 h after DNA damage induction, Supplementary Fig. 2). In contrast, at later time points (24 h after DNA damage induction), inhibition of resection clearly increases the amount of breaks detected by our assay, indicating that DNA resection can lead to an underestimation of the levels of restriction enzyme–mediated breaks. However, this can be checked in pilot experiments by using CtIP siRNAs. If required, resection-dependent effects can be minimized by short periods of incubation with the restriction enzyme, which limits the extent of DNA resection. Comparison with other methods Traditionally, the amount of restriction enzyme–mediated breaks has mostly been estimated by PCR using primers present on both sides of the break10,25. In this technique, the amount of intact DNA is measured (and not the amount of cleaved DNA). Thus, cleavage is assessed by a decrease in PCR amplification, and the quantification is only accurate for very large amounts of breaks (>30–50%). Below this cutting efficiency, the decrease in PCR signal that reflects cleavage would be below the reproducibility of real-time PCR. ligation-mediated purification, which measures a positive signal (the amount of cleaved DNA), is consequently more sensitive (Supplementary Fig. 3), and the presence of a percentage of breaks as low as 0.1% can be routinely detected. 520 | VOL.9 NO.3 | 2014 | nature protocols

Site 1

1 0.5

b 109,830,000

109,850,000

109,870,000

Site 2

0.5 0

Position (bp)

0.3 0.2 0.1 0 –0.1

Si te

–0.5

0.4

38,460,000

38,480,000

38,500,000

2

1

109,810,000

1

Position (bp) EcoRI

Si te

0 –0.5

Average log2 (strep/input) between EcoRI sites

Log2 (strep/input)

a

Log2 (strep/input)

Figure 4 | Typical experiment analyzed on tiling arrays. (a) U2OS cells expressing ER-AsiSI were subjected to ligation-mediated purification without or with DNA breaks induction (induced by a 4-h OH-Tam treatment). Pulled-down DNA and input DNA were amplified and hybridized to the Human Tiling Array 2.0R A, covering the human chromosome 1 and 6. Data obtained for two AsiSI sites are shown as the log2 ratio of pull-down sample/input sample. AsiSI sites are indicated by arrows. The meaningful signal is expected between the two EcoRI sites surrounding the AsiSI site (EcoR1 being the restriction enzyme A for this experiment; it limits the size of pulled-down fragments). The top image shows an AsiSI site efficiently cleaved, whereas the bottom image shows a site apparently uncleaved in living cells. The small vertical red lines show all EcoRI sites, and the dotted lines indicate the EcoRI sites flanking the AsiSI site. (b) For each site, the average signal obtained between the two flanking EcoRI sites can be calculated, and it represents the relative extent of cleavage. Such calculation can be made for all sites in the genome, leading to genome-wide assessment of the extent of cleavage.

38,520,000

EcoRI

The amount of sequence-specific DNA breakage has also been measured by ligation-mediated PCR (LM-PCR)15,29–31. In this technique, an asymmetrical linker is ligated to ends of genomic DNA32,33. A PCR is then performed with one primer targeting the genomic DNA of interest, and the other targeting the linker. We have found that, although LM-PCR is more sensitive than the overlapping PCR described above, it is significantly less sensitive than ligation-mediated purification (Supplementary Fig. 3). Note that in the case of I-SceI the nonpalindromic 3′ protruding end of the digested DNA prevents the linker (Fig. 2) from self-ligating, and it is possible to perform a direct PCR after the ligation step of ligation-mediated purification by using a primer in the linker and a primer in the sequence of interest. Such a ‘cohesive’ LMPCR, although slightly more efficient than the classical LM-PCR described above, is still less sensitive than ligation-mediated purification (data not shown). In addition, given that the percentage relative to inputs is calculated, ligation-mediated purification is not dependent on qPCR efficiency. This allows accurate comparison of the extent of cleavage at multiple sites within the genome and also to easily determine the absolute percentage of cleavage (Fig. 3). Unlike LM-PCR, ligation-mediated purification is compatible with genome-wide analysis of break induction (Fig. 4; ref. 13). In addition, ligation-mediated purification allows more flexibility in the choice of PCR primers, which may avoid time-consuming optimization of qPCR settings. Genome-wide techniques based on biotin labeling and streptavidin purification have recently been developed for mapping the preferred genomic localization of endogenous DSBs (produced during replication, for example)34,35. Although these techniques do not address the same questions as ligation-mediated purification, they demonstrate the growing interest in such streptavidinbased purification methodologies. Experimental design Use of alternative cleavage enzymes. We have only used AsiSI or I-SceI, which produce single-stranded protruding ends. However, this method of sequence-specific biotin labeling is probably compatible with other restriction enzymes or with other endonucleases made sequence-specific through the addition of zinc-finger nucleases36,37 or transcription activator–like effector domains38,39. The only prerequisite is that the exact sequence of the DNA end is known to allow the design of an appropriate biotinylated doublestranded oligonucleotide.

protocol


Controls for qPCR analysis. Two negative controls need to be included in ligation-mediated purification experiments: a sample without ligase demonstrates that any signal is due to a ligationdependent step (the ‘no ligase’ point in Fig. 3a), and amplification of a genomic sequence far from the restriction enzyme-induced break assesses for the specificity of the enrichment (the ‘control genomic sequence’ point in Fig. 3a). In addition, we recommend when possible to include a sample in which no break is induced in cells (without I-SceI expression in Fig. 3a, without 4-hydroxytamoxifen (OH-Tam) in Fig. 3d). An internal control, consisting of a plasmid that was previously cut in vitro by using the restriction enzyme used for in vivo DSB induction and included in all samples at the ligation step, is used to assess the efficiency of the whole procedure (internal control in Fig. 3a; please note that we divided the

percentage of input of this internal control by 20-fold for figure clarity). Moreover, by dividing the signal obtained around the in cellulo–cleaved site by this internal control, it is possible to standardize for experimental variations between the various samples. This step is useful when the amount of internal control is markedly different between the various samples (e.g., because of unequal pipetting of agarose beads). In addition, this internal control assesses the efficiency of the whole procedure, and provided that it is known that this internal control is cleaved at 100% in vitro (Steps 4–6 below), such standardization allows the determination of the absolute percentages of the extent of cleavage. The accuracy of such calculations is indicated by the fact that a 30% mix between in vitro I-SceI–digested genomic DNA and uncleaved genomic DNA is measured as 31% of the cleavage extent (Fig. 3c).

MATERIALS REAGENTS • Appropriate cell lines. In this manuscript, we have used GC92 (ref. 40) and U2OS ER-AsiSI13 cell lines (Reagent Setup). Any cell lines, strains, plants or animals in which active restriction enzymes can be expressed can be used, provided that the genome is sequenced  CRITICAL Be sure to adhere to all relevant institutional ethics and, if appropriate, animal use and care guidelines. • Internal control plasmids. We used the pPURO-HR-sub-CO-pSV40 plasmid and the pCEP4FHDAC3 as internal controls for I-SceI and AsiSI experiments, respectively. These plasmids are available from the authors upon request. Any plasmid containing a site for the restriction enzyme of interest can be used, provided that it can be specifically detected by qPCR • Endonuclease-expressing plasmid. In this manuscript, we used the pcDNA3βmycNLS-I-SceI plasmid to express the I-SceI endonuclease after transient transfection of cultured cells. This plasmid is available from the authors upon request • Gel purification kit: GenElute agarose spin columns (Sigma-Aldrich, cat. no. G2291) • Plasmid purification kit: Endofree plasmid maxi kit (Qiagen, cat. no. 12362) • Genomic DNA purification kit: DNeasy blood and tissue kit (Qiagen, cat. no. 69504) • Trizma base (Tris; Sigma, cat. no. T1503) • EDTA disodium salt dihydrate (Sigma, cat. no. E5134) • Albumin, acetylated from bovine serum (BSA; Sigma, cat. no. B8894) • UltraPure salmon sperm DNA solution (Invitrogen, cat. no. 15632-011) • T4 DNA ligase (Promega, cat. no. M180) • T4 poly kinase (Promega, cat. no. M410) • Adenosine-5′-triphosphate (ATP; Roche, cat. no. 10127523001) • Protein A–agarose (Sigma, cat. no. P7786) • Streptavidin, immobilized on Agarose CL-4B (streptavidin agarose beads; Fluka, cat. no. 85881) • SDS (Euromedex, cat. no. EU0460-B) • Triton X-100 (Sigma, cat. no. T8787) • Sodium chloride (NaCl; Sigma-Aldrich, cat. no. S3014) • UltraPure glycogen (Invitrogen, cat. no. 10814010) • Sodium acetate (Fluka, cat. no. 71180) • Ethanol (VWR, cat. no. 20821-296) • UltraPure phenol:chloroform:isoamyl alcohol (25:24:1 (vol/vol); Invitrogen, cat. no. 15593-031) ! CAUTION It is harmful if inhaled; manipulate it under a chemical hood. • Chloroform (Fluka, cat. no. 25666) • IQ SYBR Green mix (Bio-Rad, cat. no. 1708887) ! CAUTION This is a DNA intercalating agent: discard any contaminated material in a specific bin for DNA intercalating agents. • Nuclease-free water (Promega, cat. no. P119C) • EcoRI (Promega, cat. no. R601) • ScaI (Promega, cat. no. R621) • HindIII (Promega, cat. no. R604)

• I-SceI (NEB, cat. no. R0694) • AsiSI (NEB, cat. no. R0630) • Binding buffer (Reagent Setup) • Washing buffer (Reagent Setup) • TE (10 mM Tris, pH 7.5, 1 mM EDTA) Generic oligonucleotides (Eurogentec) (Reagent Setup) • Oligonucleotides for generating the I-SceI linker, as shown in Figure 2: 5′-dRbiot-CCCTATAGTGAGTCGTATTAAAGCTTGCGTTAT-3′ 5′-CGCAAGCTTTAATACGACTCACTATAGGG-3′ • Oligonucleotides for generating the AsiSI linker shown in Figure 2: 5′-dRbiot-CCCTATAGTGAGTCGTATTAAAGCTTGCGAT-3′ 5′-CGC AAGCTTTAATACGACTCACTATAGGG-3′ Custom oligonucleotides (Eurogentec) (Reagent Setup) • Oligonucleotides for qPCR quantification of sequence near the I-SceI site from GC92 cells: FW: 5′-GACTGGCACGACAGAACTGA-3′; REV: 5′-TATGGCTTCATCCCACAACA-3′ • Oligonucleotides for qPCR quantification of the internal control for I-SceI: FW: 5′-GGCCACAAGTTCAGCGTGTC-3′; REV: 5′-AAGCACTGCACG CCGTAGGT-3′ • Oligonucleotides for qPCR quantification of the control genomic sequence derived from phosphoprotein P0: FW: 5′-GGCGACCTGGAAGTCCAACT-3′; REV: 5′-CCATCAGCACCACAGCCTTC-3′ • Oligonucleotides for qPCR quantification of the internal control for AsiSI: FW: 5′-ACATTGTTGGAGCCGAAATC-3′; REV: 5′-CGCAAGGAATCGGT CAATAC-3′ EQUIPMENT • Refrigerated microcentrifuge (Eppendorf, model no. 5417R) • Spectrophotometer (NanoDrop, model no. ND-1000) • Water bath • DNA LoBind tube, 1.5 ml (Eppendorf, cat. no. 022431021) • Low-profile 96-well PCR plate (Thermo, cat. no. AB-700) • PCR sealers Microseal B film (Bio-Rad, cat. no. MSB1001) • Q-PCR instrument (Bio-Rad, model no. CFX96 real-time system) • Micropipettes (adjustable volume pipettes), 2, 10, 20 and 200 µl and 1 ml (Gilson), and filter pipette tips (ART) REAGENT SETUP Binding buffer Combine 20 mM Tris, pH 8.1, 0.1% (wt/vol) SDS, 1% (vol/vol) Triton X-100, 2 mM EDTA and 150 mM NaCl. This buffer can be stored for 1 week at 4 °C. Washing buffer Combine 50 mM Tris, pH 8.1, 0.1% (wt/vol) SDS and 150 mM NaCl. This buffer can be stored for 1 week at 4 °C. Cell lines GC92 (ref. 40) and U2OS ER-AsiSI13 cell lines can be grown and treated as described previously. GC92 cells are immortalized human fibroblasts presenting a transgene containing two I-SceI sites, which can be cut after transient transfection of an expression vector for I-SceI endonuclease (which does not cut the human genome). U2OS ER-AsiSI cells are derived from U2OS human osteosarcoma cells and express a fusion

nature protocols | VOL.9 NO.3 | 2014 | 521


protocol protein between the AsiSI enzyme (the AsiSI recognition site is present at more than 1,000 sites in the human genome) and the ligand-binding domain of the ER. In the absence of OH-Tam, AsiSI is inactive (sequestered in the cytoplasm), but in the presence of OH-Tam AsiSI is activated (relocalized into the nucleus) and it can cut its target sites. Oligonucleotide design for biotinylated double-strand linkers Order two complementary oligonucleotides that, after hybridization, create a site cohesive with DNA digested by the enzyme of interest (I-SceI or AsiSI, in the examples shown in Fig. 2b). Include a biotin on one of the oligonucleotides at the opposite end to the cohesive site. Also include a site for a restriction enzyme used to elute pulled-down DNA after purification. We used HindIII (Fig. 2), but any restriction enzyme can be used, provided that it does not cut between the DSB of interest (either in genomic DNA or in the internal control) and the primers used for the detection by qPCR. Note that ordering phosphorylated oligonucleotides, although not required, will increase the ligation efficiency. Primer design for qPCR detection Order primers that enable sequences near enzyme-mediated DSBs on the genomic DNA and on the internal control to be detected by qPCR. Also order primers to analyze a genomic sequence far from any DSB. Choose primers of 20 nt length, with a melting temperature of ~60 °C, and that amplify a sequence from 80 to 120 bp. Check that these primers function adequately by performing a standard curve with genomic DNA or the internal control (for primers detecting the internal control). For example, perform serial dilutions (1-, 3-, 10-, 30- and 100-fold) and calculate the efficiency of PCR amplification. If the efficiency obtained is below 95%, order a new set of redesigned primers. See ref. 41 for a precise description of the general rules for selecting efficient primers.

 CRITICAL For enzymes that do not cut palindromic sites (such as I-SceI), the oligonucleotide will be ligated to only one side of the break; be careful to choose primers for qPCR detection on the correct side of the break. Preparation of the double-stranded biotinylated oligonucleotide linker Hybridize the two complementary oligonucleotides by resuspending each primer at 100 µM and mixing in a microtube 20 µl of each primer, 25 µl of 1M Tris (pH 7.7) and 35 µl of H2O. Incubate the tube for 3 min at 95 °C, and then put it in a water bath at 70 °C and allow it to cool slowly to room temperature (20 °C). The biotinylated linker may be stored for months at −20 °C.  CRITICAL Throughout the procedure, use tips with filters to avoid contaminating pipettes, which would lead to high background in the final qPCR steps. Preparation of the internal control Choose a plasmid containing a site for the restriction enzyme used to induce DSBs in vivo (AsiSI or I-SceI) and that allows its specific detection by qPCR. In particular, when analyzing cleavage of a transgene (as with I-SceI), ensure that this plasmid is different from the plasmid used to make the transgenic cell line. Mix 10 µg of plasmid and 5 µl of appropriate 10× buffer in a tube and adjust the final volume to 48 µl with H2O. Add 2 µl of AsiSI or I-SceI. Incubate the tube for 1 h at 37 °C. Inactivate the enzyme by heat inactivation (15 min at 65 °C for AsiSI and I-SceI). Check that the digestion is complete by migration on an agarose gel. Store the control at −20 °C. If the digestion is not complete, purify the linear form from an agarose gel. The internal control may be stored for months at −20 °C.  CRITICAL To obtain an absolute percentage of the extent of cleavage, it is crucial to ensure that the cleavage efficiency on the internal control is 100%.

PROCEDURE Preparation of genomic DNA ● TIMING 1 h 1| Induce sequence-specific DSBs in cells, plants or animals according to your experimental strategy. In case you plan to analyze the effect of a specific treatment or an interfering RNA or a recombinant protein on the signaling and repair parameters of these sequence-specific DSB, also prepare a sample with the appropriate control. If possible, include a negative control without restriction enzyme–mediated breaks (such as –OH-Tam for ER-AsiSI in ref. 13 or in the absence of I-SceI expression vector in ref. 26 and in Fig. 3a). We classically use 1 × 106 cells to obtain ~20 µg of genomic DNA. 2| Prepare genomic DNA according to your favorite procedure. We use the DNeasy blood and tissue kit from Qiagen and prepare genomic DNA according to the manufacturer’s instructions. In some instances, we have found that treating with RNase during genomic DNA preparation (as recommended by the manufacturer) increases the yield and the quality of DNA extraction. Elute genomic DNA in 50 µl of H2O. Calculate the concentration and check the quality of genomic DNA by measuring the OD at 280 and 260 nm. Ligation of the biotinylated oligonucleotides ● TIMING 2.5 h 3| Prepare two microtubes containing the following components: Component

Amount per reaction (ml)

Final amount or concentration

Genomic DNA (10 µg µl–1)

1

1 µg

Internal control (10 pg µl–1)

1

10 pg

Biotinylated oligonucleotide (20 µM)

1

0.66 µM

T4 DNA ligase buffer with ATP (10×)

3

1×

22

—

H 2O

 CRITICAL STEP If you plan to use the genome-wide detection option in Step 21, do not include the internal control at this step.

522 | VOL.9 NO.3 | 2014 | nature protocols

protocol 4| Add 2 µl of T4 DNA ligase (3 units) to one of the tubes; the other tube will be used as a ‘no-ligase’ control and processed in parallel. Incubate both tubes at 4 °C for 2 h. 5| Inactivate the ligase by heating the samples at 65 °C for 10 min.


Streptavidin-agarose bead preparation and purification of the biotinylated fragments (before adding beads) ● TIMING ~3.5 h 6| Add 2 µl of 10× digestion buffer and 1 µl of EcoRI. Incubate the mixture for 1 h at 37 °C. During the incubation, prepare streptavidin-agarose beads (for purification) and protein A–agarose (for a preclearing step) as described in Steps 7–11 below.  CRITICAL STEP The goal of this step is to fractionate genomic DNA to a mean size between 1,000 and 5,000 bp. This step will limit the size of the genomic DNA fragments that will be pulled down with the biotinylated oligonucleotide. We typically use the 6-bp cutter EcoRI (Fig. 4), although in some instances we have used ScaI (Fig. 3). Any 6-bp cutters should work, but be careful to choose a restriction enzyme that does not cut between the DSB of interest (either in genomic DNA or in the internal control) and the primers used for detection by qPCR. 7| Pipette the equivalent of 20 µl of both dry streptavidin- and protein A–agarose beads per sample (count one extra sample in order to have enough beads). 8| Wash the beads twice with 1 ml of binding buffer 9| Resuspend the beads at 50% by adding 1 volume of binding buffer, and then add 0.2 mg ml−1 salmon sperm DNA and BSA (0.5 mg ml−1) to saturate the beads.  CRITICAL STEP If you plan to use the genome-wide detection option in Step 21, do not include salmon sperm DNA at this step. 10| Incubate the beads for 3 h at 4 °C on a rotating wheel. 11| Wash the beads once with 1 ml of binding buffer: add 1 ml of binding buffer to the beads, and centrifuge the mixture at 100g for 1 min at 4 °C; discard the supernatant and resuspend the beads at 50% (vol/vol) in binding buffer. 12| Add 950 µl of binding buffer to each sample from Step 6. Preclear the samples by adding 40 µl of prewashed protein A–agarose beads (at 50% (vol/vol)). Incubate the samples 1 h on a rotating wheel at 4 °C. 13| Centrifuge the samples at 20,000g for 10 min and transfer the supernatant to a new tube.  CRITICAL STEP Take care when pipetting the supernatant to not carry over beads, as this is a major cause of background at Step 21. 14| Collect 180 µl of each sample as input. To the remainder of each sample, add 40 µl of prewashed streptavidin-agarose beads from Step 11. Incubate the samples on a rotating wheel for 2 h (longer incubation times are possible: we usually use overnight incubations to have samples ready the next morning).  CRITICAL STEP Take care to pipette the same amount of beads per sample: we usually cut the tips with a scalpel or scissors to increase the diameter of the tip to ensure consistency of pipetting.  PAUSE POINT Store the 180-µl input samples at −20 °C until required at Step 19. Purification of biotinylated fragments (washing steps) ● TIMING 0.5–1 h 15| Wash the beads five times with 1 ml of washing buffer: add 1 ml of washing buffer to the beads, and centrifuge at 100g for 1 min at 4 °C; discard the supernatant and repeat this step five times. 16| Wash the beads twice with TE. Add 1 ml of TE to the beads and then centrifuge the tubes at 100g for 1 min at 4 °C; discard the supernatant and repeat this step twice. At the end of the last washing step, resuspend the beads in 180 µl of H2O.  CRITICAL STEP Washing steps (Steps 15 and 16) ensure that the nonspecific binding (noise) is minimal. Elution of DNA from beads ● TIMING 3–4 h 17| Digest the DNA with the restriction enzyme whose site was included in the oligonucleotide (HindIII in our experiments): Add 20 µl of 10× digestion buffer and 2 µl of enzyme. Incubate the tubes for 4 h at 37 °C. Tap the microtubes regularly during incubation to resuspend the beads and to increase the recovery yield. nature protocols | VOL.9 NO.3 | 2014 | 523

protocol  CRITICAL STEP Because of the large amount of HindIII recognition sites that are present in the biotinylated oligonucleo tides, this step is the limiting step of the whole procedure. The use of either too much or too little HindIII decreases the efficiency of the technique, leading, respectively, to inefficient digestion of the canonical HindIII sites or nonspecific digestion at noncanonical sites. 18| Centrifuge the samples (1,000g, 5 min at room temperature) to pellet the beads, and collect the supernatant in a clean tube.  CRITICAL STEP Take care not to transfer any beads, which would strongly increase the background at later steps. We recommend pipetting only 160–180 µl to ensure that no beads will be transferred. 19| Precipitate DNA (from Step 18) and inputs (from Step 14) by adding 20 µg of glycogen, 20 µl of 3M sodium acetate (pH 5.2), and 400 µl of ethanol. Centrifuge at full speed (20,000g) for 20 min at 4 °C in a microcentrifuge.


20| Wash the pellet with 70% (vol/vol) ethanol. Dry the pellet and resuspend it in 100 µl of H2O. Detection of the sequence of interest 21| The sequence of interest can be detected in two ways: by real-time PCR (option A) or by genome-wide approaches (option B). Option A allows quantitative information on one break to be obtained, whereas option B is suitable for analyzing the extent of DSB presence at multiple sites on the genome at the same time, when using a restriction enzyme generating several DSBs (such as AsiSI, as performed in ref. 13). (A) Detection by real-time PCR ● TIMING 3 h + 1 h of data analysis (i) Prepare qPCR primers (10 µM each in H2O) that enable detection of sequences near enzyme-mediated DSBs on the genomic DNA and on the internal control. Also prepare control primers (10 µM each in H2O) to analyze a genomic sequence far from any DSB. (ii) Perform (in triplicate) PCR detection of all samples (inputs, +ligase and –ligase) with the three primer pairs. Ensure to include control wells without samples to check for contamination (H2O wells). Mix the following in a 96-well plate: Component

Amount per reaction (ml)

Final amount or concentration

Primer, forward (10 µM)

1

400 nM

Primer, reverse (10 µM)

1

400 nM

12.5

1×

8

—

2.5

—

SYBR Green–containing mix (IQ Bio-Rad) (2×) Sample H 2O

Alternatively, to save money, 10-µl reactions can be prepared with 2 µl of sample. However, this results in less-consistent triplicates. ! CAUTION Because of the presence of a DNA intercalating agent (SYBR Green), discard all contaminated materials according to appropriate regulations. (iii) Use an appropriate protocol for amplification and detection by real-time PCR; a typical example from our laboratory is given below. Annealing temperature may be adapted according to the primers used. Cycle number 1 2–39

Denature

Anneal

Extend

Read fluorescence

95 °C, 3 min

—

—

—

95 °C, 15 s

60 °C, 20 s

72 °C, 30 s

72 °C, 15 s

(iv) Recover the results (as Ct, cycle threshold) as suggested by the software driving the real-time PCR device, as a Microsoft Excel file. Calculate the relative amount of sequences in each sample by using the following formula: DNA amount = 2(−Ct). You may choose to calculate 2(40−Ct), 40 being arbitrarily chosen to obtain values above 1 (if the Ct values are below 40). The 2(40) will be eliminated when calculating the percentage relative to inputs, as described in Step 21A(v). ? TROUBLESHOOTING


protocol Box 1 | Phenol/chloroform extraction ● TIMING 1 h


1. Add 200 µl of H2O to the streptavidin beads and 200 µl of phenol saturated with TE (pH 8.0). Vortex and centrifuge the mixture for 10 min at full speed (20,000g) in a microcentrifuge at room temperature. Collect the upper phase and transfer it to a new tube. 2. Re-extract the phenol phase by adding 200 µl of TE. Repeat the vortex and centrifugation steps. 3. Combine the two upper phases. Add 400 µl of chloroform/isoamyl alcohol. Vortex and centrifuge the mixture at full speed (20,000g) for 10 min in a microcentrifuge. Collect the upper phase. 4. Precipitate DNA: add 40 µl of Na-Ac 3M, pH 5.2, 900 µl of ethanol and 20 µg of glycogen. Centrifuge the mixture at full speed (20,000g) for 20 min at 4 °C in a microcentrifuge. 5. Resuspend the mixture in 100 µl of H2O. ! CAUTION Manipulate the phenol under a chemical hood. Discard any materials contaminated with phenol according to appropriate regulations.

(v) For each primer pair and each sample, calculate the percentage of input: divide the mean of the amount of DNA recovered (multiplied by 100) by the mean of the amount of DNA present in the corresponding input. Such calculations are shown in Figure 3a for a typical experiment. ? TROUBLESHOOTING (vi) Check that the signal (percentage relative to inputs in the +ligase point from samples in which DSBs were induced) is markedly higher than all negative controls (−ligase sample (no ligase in Fig. 3a), the sample without DSB induction if applicable (−I-SceI in Fig. 3a) and a genomic sequence far from any enzyme site (control genomic sequence (from the P0 phosphoprotein encoding gene) in Fig. 3a)). ? TROUBLESHOOTING (vii) Standardize for the pull-down efficiency by dividing the signals by their respective internal controls (Fig. 3b). Note that if cleavage of the internal control is 100%, the numbers obtained give the absolute percentage of cleavage. (B) Detection of microarrays ● TIMING 2 d for sample preparation and 1 week for hybridization and data analysis (i) Control the pull-down efficiency (as percentage relative to inputs) by qPCR, as described in Step 21A(i–vi) for two known cleavage sites and a DNA sequence far from any cleavage site. Check that the percentage relative to inputs is substantially higher for the known cleavage sites than for the DNA sequence far from any cleavage site. (ii) Take 8 ng of inputs and pull-down samples to perform Sequenase amplification of the DNA (as described in the Affymetrix chromatin immunoprecipitation assay protocol available at http://www.affymetrix.com). At the end of the reaction, add 20 µl of H2O to each sample.  PAUSE POINT Keep the samples frozen at −20 °C. The samples can be stored for weeks at this temperature. (iii) Take 10 µl of the reaction from Step 21B(ii) and set up PCR amplification and dUTP labeling. Follow the procedure described in the Affymetrix chromatin immunoprecipitation assay protocol, except for the PCR program. Instead use the following PCR setup: Cycle number 1 2–19 20

Denature

Anneal

Extend

95 °C, 3 min

—

—

95 °C, 30 s

45 °C, 30 s; 55 °C, 30 s

72 °C, 1 min

—

—

72 °C, 6 min

 CRITICAL STEP The number of cycles used in this PCR should be adjusted for each experiment, both in order to conserve the enrichment of the samples (checked at Step 21B(iv)) and to have enough material for hybridization (Step 21B(v)). Typically, after initial denaturation (cycle 1), 18–20 cycles are performed before the final 6-min extension at 72 °C. (iv) Purify the samples by phenol/chloroform extraction, as described in Box 1. Check by qPCR that the enrichment of positive versus negative samples is still conserved after this amplification step. (v) Purify the samples on the PrepEASE column and elute them with 40 µl of water. Measure the amount of DNA. Calculate the concentration of amplified samples by measuring the OD at 280 and 260 nm. About 9 µg of DNA is required for the subsequent step. (vi) Perform labeling and hybridization on microarray, as described in the Affymetrix chromatin immunoprecipitation assay protocol. nature protocols | VOL.9 NO.3 | 2014 | 525

protocol (vii) Use the Tiling Affymetrix Software (TAS, free software available at http://www.affymetrix.com) to perform normalization of the microarray data. Briefly, upload the .cel files within the software. Normalize the streptavidin sample against the input sample, and calculate the log2 ratio by using a bandwidth of 300 bp. The file exported as .chp can next be visualized in the Integrated Genome Browser (IGB; available at http://bioviz.org/igb/). Figure 4 shows the example of two AsiSI sites, one well cut in vivo (top panel) and the other apparently uncleaved in vivo (bottom panel). For each AsiSI site, the mean signal retrieved between the two flanking EcoRI sites (EcoRI being used as enzyme A; Fig. 1) can next be calculated in order to quantitatively compare the extent of cleavage between all AsiSI sites.

? TROUBLESHOOTING Troubleshooting advice can be found in Table 1.


Table 1 | Troubleshooting table. Step

Problem

Possible reason

Solution

21A(iv)

Inconsistent PCR triplicates

Insufficient DNA in the reaction (Ct >31)

Use more genomic DNA in the procedure

DNA of insufficient quality

Replace Step 18 by a phenol/chloroform extraction (as described in Box 1)

Signal in the H2O wells

Contamination of materials or reagents

Change reagents, wash materials and use tips with filters

Unequal inputs

Inefficient genomic DNA extraction at some sites

Add RNase during genomic DNA preparation, as recommended by the manufacturer, if applicable

Good PCR signal in the input DNA but no signal in the internal control

The ligation step did not work

Re-prepare digested plasmid and double-strand oligonucleotides. Change the ligase and make sure that the ATP is included in the ligase reaction

21A(v)

Large variations in the internal control between samples

Inconsistent amounts of streptavidin beads between samples

Ensure that streptavidin beads are well resuspended before pipetting. Cut pipette tips with a scalpel before pipetting the beads

21A(vi)

Signal not markedly higher than negative controls

Low signal (1% of input)

Increase the stringency of the washing steps (by including, for example, two extra washes with high-salt (1 M) buffer after Step 15). Ensure that the blocking step (Step 9) was adequately performed

● TIMING Day 1 Steps 1 and 2, preparation of genomic DNA: 1 h Steps 3–5, ligation of the biotinylated oligonucleotides: 2.5 h Steps 6–14, bead preparation and purification of biotinylated fragments (until the addition of streptavidin beads): ~3.5 h (2.5 h on the first day and 1 h on the following day, followed by incubation with streptavidin beads overnight) Day 2 Steps 15 and 16, purification of biotinylated fragments (washing steps): 0.5–1 h, depending on the number of samples Steps 17–20, elution of DNA from beads: 3–4 h, depending on whether a phenol/chloroform step is required Step 21A(i–iii), detection by real-time PCR: 3 h Step 21A(iv–vii), data analysis: 1 h Days 2–6 Step 21B, detection of microarrays, if applicable: 5 d Step 21B(i), control of pull-down efficiency: 3 h, carried out on day 2 Step 21B(ii–v), preparation of samples for genome-wide analyses: days 3 and 4 526 | VOL.9 NO.3 | 2014 | nature protocols

protocol


Step 21B(vi), fragmentation, labeling and hybridization on Affymetrix microarrays: days 4 and 5 Step 21B(vii), data analysis using TAS software, day 6: 2 h Box 1, phenol/chloroform extraction: 1 h ANTICIPATED RESULTS In Step 21A(v), 24–26 PCR cycles are usually required for detecting the inputs and the positive control. The number of PCR cycles required for detecting the samples is obviously dependent on the extent of cleavage, but usually ranges between 25 and 30. When this number is >30, triplicates are less consistent and the accuracy of the measurements strongly decreases. When analyzing by qPCR, we typically obtain values corresponding to ~10–40% of input for the internal control (Fig. 3a) (the efficiency of the ligation step is typically ~70%, that of the pull-down is ~80% and that of the HindIII cleavage is ~40%). The percentage relative to inputs for the control genomic sequence must be below 0.1%. If it is more than this value, the bead-preparation step (Step 12), the preclearing step (Step 13) and the washing steps (Step 15) should be adapted to increase stringency. The extent of cleavage in cells is obviously not predictable in advance, as it depends on DNA accessibility; for example, in our experiments using AsiSI, for which we analyzed 144 AsiSI sites in the same cell population, the presence of breaks varied from one site to the other from undetectable (below 1%) to nearly 100% (ref. 13) (absolute percentages were calculated after qPCR measurement and analyzed relative to the internal control, as described in the PROCEDURE). For experiments in which expression of the restriction enzyme (I-SceI for example) is dependent on transient transfection of an expression vector, such as the ones shown in Figure 3 or described ref. 26, the percentage of cleavage is largely determined by transfection efficiency: indeed, nontransfected cells will not have their I-SceI sites cleaved, and the extent of cleavage will thus decrease with transfection efficiency. It is thus crucial to optimize transfection efficiency before the experiments. In addition, it may be useful to co-transfect in each experiment an expression vector expressing a fluorescence marker and to calculate transfection efficiency by flow cytometry analysis of fluorescence.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments This work was supported by grants to D.T. from the Ligue Nationale Contre le Cancer as an ‘équipe labellisée’, from the Association pour la Recherche contre le Cancer as a ‘programme ARC’ and from the Agence Nationale pour la Recherche (project 2011 blanc SVSE8 PinGs), and to G.L. from the Association Contre le Cancer (ARC), Agence Nationale pour la Recherche (ANR-09-JCJC-0138), Cancéropôle Grand Sud-Ouest and La Fondation Recherche Innovation Thérapeutiqe Cancérologie (Fondation RITC). F.A., P.C. and C. Courilleau were supported by grants from the French Ministry of Research. We thank M. Vandromme for critical reading of the manuscript. AUTHOR CONTRIBUTIONS C. Chailleux has contributed to the development of the assay and performed experiments for this manuscript. F.A., P.C., V.D., C. Courilleau and Y.C. have contributed to the development of the procedure. G.L. conceived the procedure and has contributed to its development. D.T. conceived the procedure and wrote the manuscript. All authors commented on and edited the manuscript and figures. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. 1. Helleday, T., Petermann, E., Lundin, C., Hodgson, B. & Sharma, R.A. DNA repair pathways as targets for cancer therapy. Nat. Rev. Cancer 8, 193–204 (2008). 2. Curtin, N.J. DNA repair dysregulation from cancer driver to therapeutic target. Nat. Rev. Cancer 12, 801–817 (2012). 3. Olive, P.L. & Banath, J.P. The comet assay: a method to measure DNA damage in individual cells. Nat. Protoc. 1, 23–29 (2006). 4. Bellaiche, Y., Mogila, V. & Perrimon, N. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics 152, 1037–1044 (1999). 5. Rong, Y.S. & Golic, K.G. Gene targeting by homologous recombination in Drosophila. Science 288, 2013–2018 (2000). 6. Liang, F., Han, M., Romanienko, P.J. & Jasin, M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc. Natl. Acad. Sci. USA 95, 5172–5177 (1998).

7. Berkovich, E., Monnat, R.J. Jr. & Kastan, M.B. Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair. Nat. Cell Biol. 9, 683–690 (2007). 8. Berkovich, E., Monnat, R.J. Jr. & Kastan, M.B. Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites. Nat. Protoc. 3, 915–922 (2008). 9. Miller, K.M. et al. Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nat. Struct. Mol. Biol. 17, 1144–1151 (2010). 10. Murr, R. et al. Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat. Cell Biol. 8, 91–99 (2006). 11. Roques, C. et al. MRE11-RAD50-NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair. EMBO J. 28, 2400–2413 (2009). 12. Caron, P. et al. Cohesin protects genes against γH2AX induced by DNA double-strand breaks. PLoS Genet. 8, e1002460 (2012). 13. Iacovoni, J.S. et al. High-resolution profiling of γH2AX around DNA double-strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010). 14. Massip, L., Caron, P., Iacovoni, J.S., Trouche, D. & Legube, G. Deciphering the chromatin landscape induced around DNA double-strand breaks. Cell Cycle 9, 2963–2972 (2010). 15. Soutoglou, E. et al. Positional stability of single double-strand breaks in mammalian cells. Nat. Cell Biol. 9, 675–682 (2007). 16. Bennardo, N., Cheng, A., Huang, N. & Stark, J.M. Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair. PLoS Genet. 4, e1000110 (2008). 17. Guirouilh-Barbat, J. et al. Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells. Mol. Cell 14, 611–623 (2004). 18. Reid, L.J. et al. E3 ligase activity of BRCA1 is not essential for mammalian cell viability or homology-directed repair of double-strand DNA breaks. Proc. Natl. Acad. Sci. USA 105, 20876–20881 (2008). 19. Xie, A., Kwok, A. & Scully, R. Role of mammalian Mre11 in classical and alternative nonhomologous end joining. Nat. Struct. Mol. Biol. 16, 814–818 (2009). 20. Boboila, C. et al. Robust chromosomal DNA repair via alternative endjoining in the absence of X-ray repair cross-complementing protein 1 (XRCC1). Proc. Natl. Acad. Sci. USA 109, 2473–2478 (2012).

nature protocols | VOL.9 NO.3 | 2014 | 527


protocol 21. Fnu, S. et al. Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining. Proc. Natl. Acad. Sci. USA 108, 540–545 (2011). 22. Niida, H. et al. Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase. Genes Dev. 24, 333–338 (2010). 23. Shakya, R. et al. BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity. Science 334, 525–528 (2011). 24. Siaud, N. et al. Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains. PLoS Genet. 7, e1002409 (2011). 25. Xu, Y. et al. The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair. J. Cell Biol. 191, 31–43 (2010). 26. Courilleau, C. et al. The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks. J. Cell Biol. 199, 1067–1081 (2012). 27. Sartori, A.A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007). 28. You, Z. et al. CtIP links DNA double-strand break sensing to resection. Mol. Cell 36, 954–969 (2009). 29. Catalan, N. et al. The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch µregion. J. Immunol. 171, 2504–2509 (2003). 30. Shahar, O.D. et al. Live imaging of induced and controlled DNA doublestrand break formation reveals extremely low repair by homologous recombination in human cells. Oncogene 31, 3495–3504 (2012).


31. Fenina, M. et al. I-SceI–mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells. PLoS ONE 7, e39895 (2012). 32. Villalobos, M.J., Betti, C.J. & Vaughan, A.T. Detection of DNA doublestrand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR. Methods Mol. Biol. 314, 109–121 (2006). 33. Besaratinia, A. & Pfeifer, G.P. Measuring the formation and repair of UV damage at the DNA sequence level by ligation-mediated PCR. Methods Mol. Biol. 920, 189–202 (2012). 34. Leduc, F. et al. Genome-wide mapping of DNA strand breaks. PLoS ONE 6, e17353 (2011). 35. Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013). 36. Carroll, D., Morton, J.J., Beumer, K.J. & Segal, D.J. Design, construction and in vitro testing of zinc finger nucleases. Nat. Protoc. 1, 1329–1341 (2006). 37. Bibikova, M., Golic, M., Golic, K.G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002). 38. Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39, 359–372 (2011). 39. Piganeau, M. et al. Cancer translocations in human cells induced by zinc-finger and TALE nucleases. Genome Res. 23, 1182–1193 (2013). 40. Rass, E. et al. Role of Mre11 in chromosomal nonhomologous end joining in mammalian cells. Nat. Struct. Mol. Biol. 16, 819–824 (2009). 41. Citri, A., Pang, Z.P., Sudhof, T.C., Wernig, M. & Malenka, R.C. Comprehensive qPCR profiling of gene expression in single neuronal cells. Nat. Protoc. 7, 118–127 (2012).