Skip to main content
SpringerLink
Log in

Quantifying DNA End Resection in Human Cells

  • Protocol
  • First Online:
Homologous Recombination

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2153))

Abstract

DNA double-strand break (DSB) end resection initiates homologous recombination (HR) and is critical for genomic stability. DSB resection has been monitored indirectly in mammalian cells using detection of protein foci or BrdU foci formation, which is dependent on single-stranded DNA (ssDNA) products of resection. Here we describe a quantitative PCR (qPCR)-based assay to directly measure levels of ssDNA intermediates generated by resection at specific DSB sites in human cells, which is more quantitative and precise with respect to the extent and efficiency of resection compared with previous methods. This assay, excluding the time for making the stable cell line expressing the restriction enzyme AsiSI fused to the estrogen receptor hormone-binding domain (ER-AsiSI), can be completed within 3 days.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40:179–204. https://doi.org/10.1016/j.molcel.2010.09.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Trovesi C, Manfrini N, Falcettoni M, Longhese MP (2013) Regulation of the DNA damage response by Cyclin-dependent kinases. J Mol Biol 425(23):4756–4766. https://doi.org/10.1016/j.jmb.2013.04.013

    Article  CAS  PubMed  Google Scholar 

  3. Huertas P (2010) DNA resection in eukaryotes: deciding how to fix the break. Nat Struct Mol Biol 17:11–16. https://doi.org/10.1038/nsmb.1710

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271. https://doi.org/10.1146/annurev-genet-110410-132435

    Article  CAS  PubMed  Google Scholar 

  5. Lieber MR, Gu J, Lu H, Shimazaki N, Tsai AG (2010) Nonhomologous DNA end joining (NHEJ) and chromosomal translocations in humans. Subcell Biochem 50:279–296. https://doi.org/10.1007/978-90-481-3471-7_14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhou Y, Caron P, Legube G, Paull TT (2013) Quantitation of DNA double-strand break resection intermediates in human cells. Nucleic Acids Res 42(3):e19. https://doi.org/10.1093/nar/gkt1309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Makharashvili N, Tubbs AT, Yang SH, Wang H, Barton O, Zhou Y, Deshpande RA, Lee JH, Lobrich M, Sleckman BP, Wu X, Paull TT (2014) Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection. Mol Cell 54:1022–1033. https://doi.org/10.1016/j.molcel.2014.04.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhou Y, Lee JH, Jiang W, Crowe JL, Zha S, Paull TT (2017) Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM. Mol Cell 65:91–104. https://doi.org/10.1016/j.molcel.2016.11.004

    Article  CAS  PubMed  Google Scholar 

  9. Lee JH, Mand MR, Kao CH, Zhou Y, Ryu SW, Richards AL, Coon JJ, Paull TT (2018) ATM directs DNA damage responses and proteostasis via genetically separable pathways. Sci Signal 11:512. https://doi.org/10.1126/scisignal.aan5598

    Article  Google Scholar 

  10. Myler LR, Gallardo IF, Zhou Y, Gong F, Yang SH, Wold MS, Miller KM, Paull TT, Finkelstein IJ (2016) Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc Natl Acad Sci U S A 113:E1170–E1179. https://doi.org/10.1073/pnas.1516674113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhou Y, Paull TT (2013) DNA-dependent protein kinase regulates DNA end resection in concert with Mre11-Rad50-Nbs1 (MRN) and ataxia telangiectasia-mutated (ATM). J Biol Chem 288:37112–37125. https://doi.org/10.1074/jbc.M113.514398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Iacovoni JS, Caron P, Lassadi I, Nicolas E, Massip L, Trouche D, Legube G (2010) High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome. EMBO J 29:1446–1457. https://doi.org/10.1038/emboj.2010.38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Massip L, Caron P, Iacovoni JS, Trouche D, Legube G (2010) Deciphering the chromatin landscape induced around DNA double strand breaks. Cell Cycle 9:2963–2972. https://doi.org/10.4161/cc.9.15.12412

    Article  CAS  PubMed  Google Scholar 

  14. Stenerlow B, Karlsson KH, Cooper B, Rydberg B (2003) Measurement of prompt DNA double-strand breaks in mammalian cells without including heat-labile sites: results for cells deficient in nonhomologous end joining. Radiat Res 159:502–510

    Article  CAS  Google Scholar 

  15. Miller KM, Tjeertes JV, Coates J, Legube G, Polo SE, Britton S, Jackson SP (2010) Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nat Struct Mol Biol 17:1144–1151. https://doi.org/10.1038/nsmb.1899

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zierhut C, Diffley JF (2008) Break dosage, cell cycle stage and DNA replication influence DNA double strand break response. EMBO J 27:1875–1885. https://doi.org/10.1038/emboj.2008.111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhou Y, Paull TT (2013) DNA-dependent protein kinase regulates DNA end resection in concert with the Mre11-Rad50-Nbs1 (MRN) complex and ataxia-telangiectasia-mutated (ATM). J Biol Chem 288(52):37112–37125. https://doi.org/10.1074/jbc.M113.514398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yamane A, Resch W, Kuo N, Kuchen S, Li Z, Sun HW, Robbiani DF, McBride K, Nussenzweig MC, Casellas R (2011) Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes. Nat Immunol 12:62–69. https://doi.org/10.1038/ni.1964

    Article  CAS  PubMed  Google Scholar 

  19. Yamane A, Robbiani DF, Resch W, Bothmer A, Nakahashi H, Oliveira T, Rommel PC, Brown EJ, Nussenzweig A, Nussenzweig MC, Casellas R (2013) RPA accumulation during class switch recombination represents 5′–3’ DNA-end resection during the S-G2/M phase of the cell cycle. Cell Rep 3:138–147. https://doi.org/10.1016/j.celrep.2012.12.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Aymard F, Bugler B, Schmidt CK, Guillou E, Caron P, Briois S, Iacovoni JS, Daburon V, Miller KM, Jackson SP, Legube G (2014) Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nat Struct Mol Biol 21:366–374. https://doi.org/10.1038/nsmb.2796

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Work in the Paull laboratory is supported by the Cancer Prevention and Research Institute of Texas grant RP110465-P4.

Author information

Authors and Affiliations

  1. The Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA

    Yi Zhou & Tanya T. Paull

Authors
  1. Yi Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tanya T. Paull
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tanya T. Paull .

Editor information

Editors and Affiliations

  1. Centro Andaluz de Biologı´a Molecular, y Medicina Regenerativa, CABIMER, Universidad de Sevilla, Sevilla, Spain

    Andrés Aguilera

  2. Institut Curie, PSL Research University, CNRS, UMR3348, F-91405, Orsay, France

    Aura Carreira

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Zhou, Y., Paull, T.T. (2021). Quantifying DNA End Resection in Human Cells. In: Aguilera, A., Carreira, A. (eds) Homologous Recombination. Methods in Molecular Biology, vol 2153. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0644-5_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0644-5_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0643-8

  • Online ISBN: 978-1-0716-0644-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation