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.
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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.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Controlling the levels of enzyme-mediated DSBs for a correct interpretation of ChIP data.
U2OS ER-AsiSI cells were treated for 4 hours with OH-Tam to induce AsiSI-mediated cleavage, in the presence of ATM or DNA-PK inhibitors (20μM Ku55933 and 2μM Nu 7441 respectively, added an hour previous OH-tam treatment, as indicated. a) Chromatin was prepared and analysed by chromatin immunoprecipitation using γH2AX antibodies. ChIP efficiency (as percent of input) was measured by qPCR at the vicinity of two AsiSI sites. Note the strong increase in the ChIP signal in the presence of DNA-PK inhibitor, which could be wrongly interpreted as increased DNA damage signalling upon DNA-PK inhibition. b) Genomic DNA was prepared and the amounts of AsiSI-cohesive breaks at these two sites were analysed by Ligation-Mediated Purification. Note the strong increase in the amount of breaks upon DNAPK inhibitor treatment, which indicates that results shown in a) cannot be interpreted.
a) U2OS ER-AsiSI cells were transfected with a siRNA against CtIP. 52 hours following transfection, total RNA was prepared, retrotranscribed using random primers and levels of CtIP cDNA relative to ribosomal phosphoprotein P0 were assessed by qPCR. b) Same as in a), except that 4OHT was added 48h following transfection to induce AsiSI digestion as described in ref 13. 4 and 24 hours later, as indicated, genomic DNA was prepared and the amounts of AsiSI-cohesive breaks were quantified at two AsiSI sites by Ligation-Mediated Purification. Note that DNA resection leads to the underestimation of the presence of breaks 24 hours, but not 4 hours, following break induction.
a) Left panel: Results obtained by Ligation-Mediated Purification (this experiment is also shown in Figure 3b). Right panel: Extent of cleavage in the same samples measured by qPCR with primers on each side of the break (5'-CACTATAGGGAGACCCAAGCTG and 5'-CTGGTCGGCGATTAGCGACT). Errors bars represent standard deviation of the qPCR measurements. The % of cleavage is given by the decrease in PCR efficiency (represented by the arrow). Note that the variations between two identical samples (the +I-SceI sample and the no-ligase sample which should give the same signal since they are produced from the same genomic DNA) are higher than variations between different genomic DNA. Thus, the decrease observed in the +I-SceI sample is not significant, and cleavage efficiency in this experiment is below the detection level of this method. b) Left panel: Results obtained by Ligation-Mediated Purification as shown in Figure 3c, but with a log scale. Right panel: a similar standard curve of I-SceI-digested versus uncleaved genomic DNA was analysed by LM-PCR as described in 30, 31 except that genomic DNA was treated by T4 DNA polymerase following I-SceI-mediated digestion to obtain blunt ends. Quantification of ligation products was performed by qPCR using the following primers: 5'-GGGAGATCTGAATTCCCCTA and 5'-GTCGGCGATTAGCGACTTCT. Note that the sensitivity of ligation-mediated purification is about 10-fold higher than that of LMPCR.
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Chailleux, C., Aymard, F., Caron, P. et al. Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification. Nat Protoc 9, 517–528 (2014) doi:10.1038/nprot.2014.031
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