Abstract
Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily and specifically measured by detecting the 24–32 nucleotide products of the dual-incision reaction. This chapter describes several methods for detection of repair of a specific lesion in closed-circular DNA. As a model lesion, we use the well-repaired 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Three methods are given for analysis of repair. One is to incorporate a radioactive label internally near the lesion and measure excision by detecting radioactive excised oligomers. Two other methods use DNA that is not internally labeled so that it can be stored and used when convenient. The first method for detection of repair of such unlabeled DNA is to detect excision products with a labeled complementary oligonucleotide by Southern blot hybridization. The second method is to 3′- endlabel the excised oligonucleotide directly with radiolabeled dNTP and a DNA polymerase, using a complementary oligonucleotide with a 5′-overhang that serves as a template. This protocol is fast and sensitive, but relies on accurate foreknowledge of the site of 3′-incision for the particular lesion being used.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lindahl, T. and Wood, R. D. (1999) Quality control by DNA repair. Science 286, 1897–1905.
Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A., and Ellenberger, T. (2005) DNA Repair and Mutagenesis, American Society for Microbiology Press, Washington, D.C.
Mitchell, J. R., Hoeijmakers, J. H., and Niedernhofer, L. J. (2003) Divide and conquer: nucleotide excision repair battles cancer and ageing. Curr. Opin. Cell Biol. 15, 232–240.
Huang, J. C., Svoboda, D. L., Reardon, J. T., and Sancar, A. (1992) Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5′ and the 6th phosphodiester bond 3′ to the photodimer. Proc. Natl. Acad. Sci. USA 89, 3664–3668.
Moggs, J. G., Yarema, K. J., Essigmann, J. M., and Wood, R. D. (1996) Analysis of incision sites produced by human cell extracts and purified proteins during nucleotide excision repair of a 1,3-intrastrand d(GpTpG)-cisplatin adduct. J. Biol. Chem. 271, 7177–7186.
Wood, R. D., Araújo, S. J., Ariza, R. R., et al. (2000) DNA damage recognition and nucleotide excision repair in mammalian cells. Cold Spring Harbor Symp. Quant. Biol. 65, 173–182.
de Boer, J. and Hoeijmakers, J. H. J. (2000) Nucleotide excision repair and human syndromes. Carcinogenesis 21, 453–460.
Petit, C. and Sancar, A. (1999) Nucleotide excision repair: from E. coli to man. Biochimie 81, 15–25.
Hess, M. T., Schwitter, U., Petretta, M., Giese, B., and Naegeli, H. (1997) Bipartite substrate discrimination by human nucleotide excision-repair. Proc. Natl. Acad. Sci. USA 94, 6664–6669.
Huang, J. C. and Sancar, A. (1994) Determination of minimum substrate size for human excinuclease. J. Biol. Chem. 269, 19,034–19,040.
Mu, D. and Sancar, A. (1997) DNA excision-repair assays. Prog. Nucleic Acids Res. Mol. Biol. 56, 63–81.
Yarema, K. J. and Essigmann, J. M. (1995) Evaluation of the genetic effects of defined DNA lesions formed by DNA-damaging agents. Methods 7, 133–146.
Sugasawa, K., Masutani, C., and Hanaoka, F. (1993) Cell-free repair of UV-damaged Simian virus-40 chromosomes in human cell-extracts 1. Development of a cell-free system detecting excision repair of UV-irradiated SV40 chromosomes. J. Biol. Chem. 268, 9098–9104.
Wang, Z., Wu, X., and Friedberg, E. C. (1991) Nucleotide excision repair of DNA by human cell extracts is suppressed in reconstituted nucleosomes. J. Biol. Chem. 266, 22,472–22,478.
Ura, K., Araki, M., Saeki, H., et al. (2001) ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes. EMBO J. 20, 2004–2014.
Hara, R. and Sancar, A. (2002) The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle. Mol. Cell Biol. 22, 6779–6787.
Frit, P., Kwon, K., Coin, F., et al. (2002) Transcriptional activators stimulate DNA repair. Mol. Cell 10, 1391–1401.
Gaillard, P.-H. L., Moggs, J. G., Roche, D. M. J., et al. (1997) Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair. EMBO J. 16, 6282–6289.
Kuraoka, I., Bender, C., Romieu, A., Cadet, J., Wood, R. D., and Lindahl, T. (2000) Removal of oxygen free-radical induced 5′,8 purine cyclodeoxynucleosides from DNA by the nucleotide excision repair pathway in human cells. Proc. Natl. Acad. Sci. USA 97, 3832–3837.
Sugasawa, K., Okamoto, T., Shimizu, Y., Masutani, C., Iwai, S., and Hanaoka, F. (2001) A multistep damage recognition mechanism for global genomic nucleotide excision repair. Genes Dev. 15, 507–521.
Araújo, S. J., Tirode, F., Coin, F., et al. (2000) Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH and modulation by CAK. Genes Dev. 14, 349–359.
Araújo, S. J., Nigg, E. A., and Wood, R. D. (2001) Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a pre-assembled repairosome. Mol. Cell Biol. 21, 2281–2291.
Riedl, T., Hanaoka, F., and Egly, J. M. (2003) The comings and goings of nucleotide excision repair factors on damaged DNA. EMBO J. 22, 5293–5303.
Zamble, D. B., Mu, D., Reardon, J. T., Sancar, A., and Lippard, S. J. (1996) Repair of cisplatin-DNA adducts by the mammalian excision nuclease. Biochemistry 35, 10,004–10,013.
Shivji, M. K. K., Ferrari, E., Ball, K., Hübscher, U., and Wood, R. D. (1998) Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1. Oncogene 17, 2827–2838.
Sayers, J. (1996) Viral polymerase-associated 5′→3′-exonucleases: expression, purification, and uses. Methods Enzymol. 275, 227–238.
Yarema, K. J., Lippard, S. J., and Essigmann, J. M. (1995) Mutagenic and genotoxic effects of DNA-adducts formed by the anticancer drug cis-diamminedichloroplatinum(II). Nucleic Acids Res. 23, 4066–4072.
O’Donovan, A., Davies, A. A., Moggs, J. G., West, S. C., and Wood, R. D. (1994) XPG endonuclease makes the 3′incision in human DNA nucleotide excision repair. Nature 371, 432–435.
Yang, G., Lin, T., Karam, J., and Konigsberg, W. H. (1999) Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation. Biochemistry 38, 8094–8101.
Ausubel, F. M., Brent, R., Kingston, R. E., et al. (1989) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience, New York, NY.
Sijbers, A. M., de Laat, W. L., Ariza, R. R., et al. (1996) Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease. Cell 86, 811–822.
Acknowledgments
We thank the past members of our laboratory for discussions and contributions to these procedures, Kevin Yarema and John Essigmann for instruction in preparation of oligonucleotides modified with cisplatin, Jon Sayers for T5 exonuclease, and W. Konigsberg for procedures regarding preparation of RB69 DNA polymerase.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Shivji, M.K.K., Moggs, J.G., Kuraoka, I., Wood, R.D. (2006). Assaying for the Dual Incisions of Nucleotide Excision Repair Using DNA with a Lesion at a Specific Site. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 314. Humana Press. https://doi.org/10.1385/1-59259-973-7:435
Download citation
DOI: https://doi.org/10.1385/1-59259-973-7:435
Publisher Name: Humana Press
Print ISBN: 978-1-58829-513-2
Online ISBN: 978-1-59259-973-8
eBook Packages: Springer Protocols