Skip to main content
SpringerLink
Log in

Construction of Plasmids Containing Site-Specific DNA Interstrand Cross-Links for Biochemical and Cell Biological Studies

  • Protocol
  • First Online:
DNA Repair Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 920))

Abstract

Plasmids containing a site-specific DNA interstrand cross-link (ICL) are invaluable tools for the investigation of ICL repair pathways at the biochemical and cellular level. We describe a procedure for preparation of plasmid DNA substrates containing a single ICL at a specific site. The procedure is versatile, leads to reliable yields of pure DNA substrate, and is suitable for the incorporation of virtually any type of DNA lesion into plasmids.

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 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

References

  1. Noll DM, Mason TM, Miller PS (2006) Formation and repair of interstrand cross-links in DNA. Chem Rev 106:277–301

    Article  PubMed  CAS  Google Scholar 

  2. Schärer OD (2005) DNA interstrand crosslinks: natural and drug-induced DNA adducts that induce unique cellular responses. Chembiochem 6:27–32

    Article  PubMed  Google Scholar 

  3. Guainazzi A, Schärer OD (2010) Using synthetic DNA interstrand crosslinks to elucidate repair pathways and identify new therapeutic targets for cancer chemotherapy. Cell Mol Life Sci 67:3683–3697

    Article  PubMed  CAS  Google Scholar 

  4. Knipscheer P, Räschle M, Smogorzewska A, Enoiu M, Ho TV, Schärer OD, Elledge SJ, Walter JC (2009) The fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair. Science 326:1698–1701

    Article  PubMed  CAS  Google Scholar 

  5. Räschle M, Knipsheer P, Enoiu M, Angelov T, Sun J, Griffith JD, Ellenberger TE, Schärer OD, Walter JC (2008) Mechanism of replication-coupled DNA interstrand crosslink repair. Cell 134:969–980

    Article  PubMed  Google Scholar 

  6. Zhang N, Lu X, Zhang X, Peterson CA, Legerski RJ (2002) hMutSbeta is required for the recognition and uncoupling of psoralen interstrand cross-links in vitro. Mol Cell Biol 22:2388–2397

    Article  PubMed  CAS  Google Scholar 

  7. Shen X, Do H, Li Y, Chung WH, Tomasz M, de Winter JP, Xia B, Elledge SJ, Wang W, Li L (2009) Recruitment of fanconi anemia and breast cancer proteins to DNA damage sites is differentially governed by replication. Mol Cell 35:716–723

    Article  PubMed  CAS  Google Scholar 

  8. Shen X, Jun S, O’Neal LE, Sonoda E, Bemark M, Sale JE, Li L (2006) REV3 and REV1 play major roles in recombination-independent repair of DNA interstrand cross-links mediated by monoubiquitinated proliferating cell nuclear antigen (PCNA). J Biol Chem 281:13869–13872

    Article  PubMed  CAS  Google Scholar 

  9. Wang X, Peterson CA, Zheng H, Nairn RS, Legerski RJ, Li L (2001) Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair. Mol Cell Biol 21:713–720

    Article  PubMed  CAS  Google Scholar 

  10. Zheng H, Wang X, Warren AJ, Legerski RJ, Nairn RS, Hamilton JW, Li L (2003) Nucleotide excision repair- and polymerase eta-mediated error-prone removal of mitomycin C interstrand cross-links. Mol Cell Biol 23:754–761

    Article  PubMed  CAS  Google Scholar 

  11. Hofr C, Brabec V (2001) Thermal and thermodynamic properties of duplex DNA containing site-specific interstrand cross-link of antitumor cisplatin or its clinically ineffective trans isomer. J Biol Chem 276:9655–9661

    Article  PubMed  CAS  Google Scholar 

  12. Angelov T, Guainazzi A, Schärer OD (2009) Generation of DNA interstrand cross-links by post-synthetic reductive amination. Org Lett 11:661–664

    Article  PubMed  CAS  Google Scholar 

  13. Guainazzi A, Campbell AJ, Angelov T, Simmerling C, Schärer OD (2010) Synthesis and molecular modeling of a nitrogen mustard DNA interstrand crosslink. Chemistry 16:12100–12103

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the New York State Office of Science and Technology and Academic Research NYSTAR (C040069), the Association for International Cancer Research (AICR, Nr. 00-225), the Swiss Cancer League (OCS-01413-080-2003), the National Institutes of Health (GM080454 and CA092584 to O.D.S. and GM62267, HL098316, and GM80676 to J.C.W.), and the American Cancer Society (postdoctoral fellowship PF-10-146-01-DMC to D.T.L.).

Author information

Authors and Affiliations

  1. Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland

    Milica Enoiu

  2. Department of Pharmacological Sciences, Stony Brook University, Stony Brook, USA

    The Vinh Ho

  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    David T. Long

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA

    Johannes C. Walter

  5. Stony Brook University, Stony Brook, NY, USA

    Orlando D. Schärer

Authors
  1. Milica Enoiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. The Vinh Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David T. Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johannes C. Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Orlando D. Schärer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Orlando D. Schärer .

Editor information

Editors and Affiliations

  1. , Department of Molecular Biology, Aarhus University, C.F Møllers Allé bygning 1130, Århus C, 8000, Denmark

    Lotte Bjergbæk

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Enoiu, M., Ho, T.V., Long, D.T., Walter, J.C., Schärer, O.D. (2012). Construction of Plasmids Containing Site-Specific DNA Interstrand Cross-Links for Biochemical and Cell Biological Studies. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-998-3_15

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-997-6

  • Online ISBN: 978-1-61779-998-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation