Skip to main content
SpringerLink
Log in

Methods for Studying DNA Single-Strand Break Repair and Signaling in Xenopus laevis Egg Extracts

  • Protocol
  • First Online:
DNA Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1999))

Abstract

DNA single-strand breaks (SSBs) are the most common type of DNA lesions as they are generated approximately 10,000 times per mammalian cell each day. Unrepaired SSBs compromise DNA replication and transcription programs, leading to genome instability, and have been implicated in many diseases including cancer. In this chapter, we introduce methods to study the ATR-Chk1 DNA damage response (DDR) pathway and DNA repair pathway in response to a site-specific, defined SSB plasmid in Xenopus laevis egg extracts. This experimental system can be applied in future studies to reveal many aspects of the molecular mechanisms of SSB repair and signaling in eukaryotes.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Caldecott KW (2008) Single-strand break repair and genetic disease. Nat Rev Genet 9(8):619–631. https://doi.org/10.1038/nrg2380

    Article  PubMed  Google Scholar 

  2. Yan S, Sorrell M, Berman Z (2014) Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress. Cell Mol Life Sci 71(20):3951–3967. https://doi.org/10.1007/s00018-014-1666-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40(2):179–204. https://doi.org/10.1016/j.molcel.2010.09.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tubbs A, Nussenzweig A (2017) Endogenous DNA damage as a source of genomic instability in cancer. Cell 168(4):644–656. https://doi.org/10.1016/j.cell.2017.01.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lin Y, Bai L, Cupello S, Hossain MA, Deem B, McLeod M, Raj J, Yan S (2018) APE2 promotes DNA damage response pathway from a single-strand break. Nucleic Acids Res 46(5):2479–2494. https://doi.org/10.1093/nar/gky020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nassour J, Martien S, Martin N, Deruy E, Tomellini E, Malaquin N, Bouali F, Sabatier L, Wernert N, Pinte S, Gilson E, Pourtier A, Pluquet O, Abbadie C (2016) Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells. Nat Commun 7:10399. https://doi.org/10.1038/ncomms10399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Walter J, Sun L, Newport J (1998) Regulated chromosomal DNA replication in the absence of a nucleus. Mol Cell 1(4):519–529

    Article  CAS  PubMed  Google Scholar 

  8. Van C, Yan S, Michael WM, Waga S, Cimprich KA (2010) Continued primer synthesis at stalled replication forks contributes to checkpoint activation. J Cell Biol 189(2):233–246. https://doi.org/10.1083/jcb.200909105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kumagai A, Shevchenko A, Shevchenko A, Dunphy WG (2010) Treslin collaborates with TopBP1 in triggering the initiation of DNA replication. Cell 140(3):349–359. https://doi.org/10.1016/j.cell.2009.12.049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wallace BD, Berman Z, Mueller GA, Lin Y, Chang T, Andres SN, Wojtaszek JL, DeRose EF, Appel CD, London RE, Yan S, Williams RS (2017) APE2 Zf-GRF facilitates 3′–5′ resection of DNA damage following oxidative stress. Proc Natl Acad Sci U S A 114(2):304–309. https://doi.org/10.1073/pnas.1610011114

    Article  CAS  PubMed  Google Scholar 

  11. DeStephanis D, McLeod M, Yan S (2015) REV1 is important for the ATR-Chk1 DNA damage response pathway in Xenopus egg extracts. Biochem Biophys Res Commun 460(3):609–615. https://doi.org/10.1016/j.bbrc.2015.03.077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yan S, Willis J (2013) WD40-repeat protein WDR18 collaborates with TopBP1 to facilitate DNA damage checkpoint signaling. Biochem Biophys Res Commun 431(3):466–471. https://doi.org/10.1016/j.bbrc.2012.12.144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bai L, Michael WM, Yan S (2014) Importin beta-dependent nuclear import of TopBP1 in ATR-Chk1 checkpoint in Xenopus egg extracts. Cell Signal 26(5):857–867. https://doi.org/10.1016/j.cellsig.2014.01.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Yan S (2015) Teaching and learning in a Xenopus research lab. Lab Anim (NY) 44(8):327. https://doi.org/10.1038/laban.817

    Article  Google Scholar 

  15. Costanzo V, Robertson K, Bibikova M, Kim E, Grieco D, Gottesman M, Carroll D, Gautier J (2001) Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication. Mol Cell 8(1):137–147

    Article  CAS  PubMed  Google Scholar 

  16. Dupre A, Boyer-Chatenet L, Gautier J (2006) Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex. Nat Struct Mol Biol 13(5):451–457. https://doi.org/10.1038/nsmb1090

    Article  CAS  PubMed  Google Scholar 

  17. You Z, Shi LZ, Zhu Q, Wu P, Zhang YW, Basilio A, Tonnu N, Verma IM, Berns MW, Hunter T (2009) CtIP links DNA double-strand break sensing to resection. Mol Cell 36(6):954–969. https://doi.org/10.1016/j.molcel.2009.12.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Raschle M, Knipscheer P, Enoiu M, Angelov T, Sun J, Griffith JD, Ellenberger TE, Scharer OD, Walter JC (2008) Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134(6):969–980. https://doi.org/10.1016/j.cell.2008.08.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ben-Yehoyada M, Wang LC, Kozekov ID, Rizzo CJ, Gottesman ME, Gautier J (2009) Checkpoint signaling from a single DNA interstrand crosslink. Mol Cell 35(5):704–715. https://doi.org/10.1016/j.molcel.2009.08.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lebofsky R, Takahashi T, Walter JC (2009) DNA replication in nucleus-free Xenopus egg extracts. Methods Mol Biol 521:229–252. https://doi.org/10.1007/978-1-60327-815-7_13

    Article  CAS  PubMed  Google Scholar 

  21. Cupello S, Richardson C, Yan S (2016) Cell-free Xenopus egg extracts for studying DNA damage response pathways. Int J Dev Biol 60(7–8–9):229–236. https://doi.org/10.1387/ijdb.160113sy

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Willis J, DeStephanis D, Patel Y, Gowda V, Yan S (2012) Study of the DNA damage checkpoint using Xenopus egg extracts. J Vis Exp (69):e4449. https://doi.org/10.3791/4449

Download references

Acknowledgments

The Yan lab was supported, in part, by funds from University of North Carolina at Charlotte (Duke Energy Endowment Special Initiatives Fund and Faculty Research Grants) and grants from the National Institute of General Medical Sciences (R15GM101571 and R15GM114713) and the National Cancer Institute (R01CA225637) in the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA

    Yunfeng Lin, Anh Ha & Shan Yan

Authors
  1. Yunfeng Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anh Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shan Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shan Yan .

Editor information

Editors and Affiliations

  1. Indiana University – Purdue University, Indianapolis, IN, USA

    Lata Balakrishnan

  2. Department of Biological Sciences, University of South Carolina, Columbia, SC, USA

    Jason A. Stewart

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lin, Y., Ha, A., Yan, S. (2019). Methods for Studying DNA Single-Strand Break Repair and Signaling in Xenopus laevis Egg Extracts. In: Balakrishnan, L., Stewart, J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9500-4_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9500-4_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9499-1

  • Online ISBN: 978-1-4939-9500-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation