Abstract
Cells have evolved DNA repair mechanisms to maintain their genetic information unaltered and a DNA damage response pathway that coordinates DNA repair with several cellular events. Despite a clear role for DNA damage in the form of DNA double-strand breaks (DSBs) in several cellular processes, the most commonly used methods to detect DNA lesions are indirect, and rely on antibody-based recognition of DNA damage-associated factors, leaving several important questions unanswered. Differently, here we describe DNA damage In situ ligation followed by Proximity Ligation Assay (DI-PLA), that allows sensitive detection of physical DSBs in fixed cells, through direct labeling of the DSBs with biotinylated oligonucleotides, and subsequent signal amplification by PLA between biotin and a partner protein in the proximity of the DNA break.
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18 April 2019
Work in Fabrizio d’Adda di Fagagna’s laboratory is supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Cariplo Foundation (grant 2010.0818 and 2014-0812), Fondazione Telethon (GGP12059 and GGP17111), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331).
References
Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461:1071–1078. https://doi.org/10.1038/nature08467
Hoeijmakers JH (2009) DNA damage, aging, and cancer. N Engl J Med 361:1475–1485. https://doi.org/10.1056/NEJMra0804615
Schneider L, Pellegatta S, Favaro R et al (2013) DNA damage in mammalian neural stem cells leads to astrocytic differentiation mediated by BMP2 signaling through JAK-STAT. Stem Cell Reports 1:123–138. https://doi.org/10.1016/j.stemcr.2013.06.004
Mosteiro L, Pantoja C, Alcazar N et al (2016) Tissue damage and senescence provide critical signals for cellular reprogramming in vivo. Science 354(80):aaf4445. https://doi.org/10.1126/science.aaf4445
Vitelli V, Galbiati A, Iannelli F et al (2017) Recent advancements in DNA damage transcription crosstalk and high-resolution mapping of DNA breaks. Annu Rev Genomics Hum Genet 18:87–113
Kim JA, Kruhlak M, Dotiwala F et al (2007) Heterochromatin is refractory to γH2AX modification in yeast and mammals. J Cell Biol 178:209–218. https://doi.org/10.1083/jcb.200612031
Schneider L, Fumagalli M, d’Adda di Fagagna F (2012) Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency. Cell Death Differ 19:582–591. https://doi.org/10.1038/cdd.2011.129
Soutoglou E, Misteli T (2008) Activation of the cellular DNA damage response in the absence of DNA lesions. Science 320(80):1507–1510. https://doi.org/10.1126/science.1159051.Activation
Shmuel A (1992) Identification of programmed cell death in situ. Cell 119:493–501. https://doi.org/10.1083/jcb.119.3.493
Olive PL, Wlodek D, Banáth JP (1991) DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Res 51(17):4671–4676
Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1:23–29
Söderberg O, Gullberg M, Jarvius M et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000. https://doi.org/10.1038/nmeth947
Galbiati A, Beausèjour C, d’Adda di Fagagna F (2017) A novel single-cell method provides direct evidence of persistent DNA damage in senescent cells and aged mammalian tissues. Aging Cell 16:422–427. https://doi.org/10.1111/acel.12573
Carpenter AE, Jones TR, Lamprecht MR et al (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100. https://doi.org/10.1186/gb-2006-7-10-r100
Acknowledgments
Work in Fabrizio d’Adda di Fagagna’s laboratory is supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Cariplo Foundation (grant 2010.0818 and 2014-0812), Fondazione Telethon (GGP12059 and GGP17111), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), the Italian Ministry of Education Universities and Research EPIGEN Project, a European Research Council advanced grant (322726), AriSLA (project “DDRNA and ALS”), AIRC Special Program 5 per mille metastases Project n 21091, AMANDA project Accordo Quadro Regione Lombardia–CNR, and flagship progetto InterOmics.
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Galbiati, A., d’Adda di Fagagna, F. (2019). DNA Damage In Situ Ligation Followed by Proximity Ligation Assay (DI-PLA). In: Demaria, M. (eds) Cellular Senescence. Methods in Molecular Biology, vol 1896. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8931-7_2
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DOI: https://doi.org/10.1007/978-1-4939-8931-7_2
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