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.
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Noll DM, Mason TM, Miller PS (2006) Formation and repair of interstrand cross-links in DNA. Chem Rev 106:277–301
Schärer OD (2005) DNA interstrand crosslinks: natural and drug-induced DNA adducts that induce unique cellular responses. Chembiochem 6:27–32
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
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
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
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
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
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
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
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
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
Angelov T, Guainazzi A, Schärer OD (2009) Generation of DNA interstrand cross-links by post-synthetic reductive amination. Org Lett 11:661–664
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
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.).
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© 2012 Springer Science+Business Media New York
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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
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DOI: https://doi.org/10.1007/978-1-61779-998-3_15
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-997-6
Online ISBN: 978-1-61779-998-3
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