Skip to main content
SpringerLink
Log in

DNA Damage Foci on Metaphase Chromosomes

  • Protocol
  • First Online:
Chromosome Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2519))

Abstract

After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.

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

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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

Similar content being viewed by others

References

  1. Kato TA, Nagasawa H, Weil MM, Little JB, Bedford JS (2006) Levels of gamma-H2AX Foci after low-dose-rate irradiation reveal a DNA DSB rejoining defect in cells from human ATM heterozygotes in two at families and in another apparently normal individual. RadiatRes 166(3):443–453

    CAS  Google Scholar 

  2. Genet SC, Fujii Y, Maeda J, Kaneko M, Genet MD, Miyagawa K, Kato TA (2012) Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time and temperature dependent manner. J Cell Physiol. https://doi.org/10.1002/jcp.24302

  3. Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146(5):905–915

    Article  CAS  Google Scholar 

  4. Rothkamm K, Lobrich M (2003) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc Natl Acad Sci U S A 100(9):5057–5062

    Article  CAS  Google Scholar 

  5. Cornforth MN, Bedford JS (1983) X-ray-induced breakage and rejoining of human interphase chromosomes. Science 222(4628):1141–1143

    Article  CAS  Google Scholar 

  6. Schultz LB, Chehab NH, Malikzay A, Halazonetis TD (2000) p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol 151(7):1381–1390. https://doi.org/10.1083/jcb.151.7.1381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Maser RS, Monsen KJ, Nelms BE, Petrini JH (1997) hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol Cell Biol 17(10):6087–6096. https://doi.org/10.1128/mcb.17.10.6087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Haaf T, Golub EI, Reddy G, Radding CM, Ward DC (1995) Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A 92(6):2298–2302. https://doi.org/10.1073/pnas.92.6.2298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gatei M, Young D, Cerosaletti KM, Desai-Mehta A, Spring K, Kozlov S, Lavin MF, Gatti RA, Concannon P, Khanna K (2000) ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nat Genet 25(1):115–119. https://doi.org/10.1038/75508

    Article  CAS  PubMed  Google Scholar 

  10. Davalos AR, Kaminker P, Hansen RK, Campisi J (2004) ATR and ATM-dependent movement of BLM helicase during replication stress ensures optimal ATM activation and 53BP1 focus formation. Cell Cycle 3(12):1579–1586. https://doi.org/10.4161/cc.3.12.1286

    Article  CAS  PubMed  Google Scholar 

  11. Gatei M, Zhou BB, Hobson K, Scott S, Young D, Khanna KK (2001) Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies. J Biol Chem 276(20):17276–17280. https://doi.org/10.1074/jbc.M011681200

    Article  CAS  PubMed  Google Scholar 

  12. Garcia-Higuera I, Taniguchi T, Ganesan S, Meyn MS, Timmers C, Hejna J, Grompe M, D’Andrea AD (2001) Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell 7(2):249–262. https://doi.org/10.1016/s1097-2765(01)00173-3

    Article  CAS  PubMed  Google Scholar 

  13. Yuan SS, Lee SY, Chen G, Song M, Tomlinson GE, Lee EY (1999) BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo. Cancer Res 59(15):3547–3551

    CAS  PubMed  Google Scholar 

  14. Siddique MA, Nakanishi K, Taniguchi T, Grompe M, D’Andrea AD (2001) Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells. Exp Hematol 29(12):1448–1455. https://doi.org/10.1016/s0301-472x(01)00754-8

    Article  CAS  PubMed  Google Scholar 

  15. Franchitto A, Pichierri P (2002) Bloom’s syndrome protein is required for correct relocalization of RAD50/MRE11/NBS1 complex after replication fork arrest. J Cell Biol 157(1):19–30. https://doi.org/10.1083/jcb.200110009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stewart GS, Wang B, Bignell CR, Taylor AM, Elledge SJ (2003) MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421(6926):961–966. https://doi.org/10.1038/nature01446

    Article  CAS  PubMed  Google Scholar 

  17. Wang B, Matsuoka S, Carpenter PB, Elledge SJ (2002) 53BP1, a mediator of the DNA damage checkpoint. Science 298(5597):1435–1438. https://doi.org/10.1126/science.1076182

    Article  CAS  PubMed  Google Scholar 

  18. Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, Eckhaus M, Camerini-Otero RD, Tessarollo L, Livak F, Manova K, Bonner WM, Nussenzweig MC, Nussenzweig A (2002) Genomic instability in mice lacking histone H2AX. Science 296(5569):922–927

    Article  CAS  Google Scholar 

  19. Chan KL, Palmai-Pallag T, Ying S, Hickson ID (2009) Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol 11(6):753–760. https://doi.org/10.1038/ncb1882

    Article  CAS  PubMed  Google Scholar 

  20. Kato TA, Okayasu R, Bedford JS (2009) Signatures of DNA double strand breaks produced in irradiated G1 and G2 cells persist into mitosis. J Cell Physiol 219(3):760–765. https://doi.org/10.1002/jcp.21726

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA

    Takamitsu A. Kato

Authors
  1. Takamitsu A. Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Takamitsu A. Kato .

Editor information

Editors and Affiliations

  1. Division of Diagnostic Imaging, Department of Radiology, Japan Labour Health and Safety Organization, Tokyo Rosai Hospital, Ohta-ku, Tokyo, Japan

    Eisuke Gotoh

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kato, T.A. (2023). DNA Damage Foci on Metaphase Chromosomes. In: Gotoh, E. (eds) Chromosome Analysis. Methods in Molecular Biology, vol 2519. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2433-3_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2433-3_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2432-6

  • Online ISBN: 978-1-0716-2433-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation