Abstract
Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a source of mutations. Organisms have developed an enzymatic means of repairing abasic sites in DNA that generally involves a DNA repair pathway that is initiated by a repair protein creating a phosphodiester break (“nick”) adjacent to the site of base loss. Here we describe a method for analyzing the manner in which repair endonucleases differ in the way they create nicks in DNA and how to distinguish between them using cellular crude extracts.
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References
Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, D.C.
Lindahl, T. and Nyberg, B. (1972) Rate of depurination of native deoxyribonucleic acid. Biochemistry 11, 3610–3618.
Loeb, L. A. and Preston B. D. (1986) Mutagenesis by apurinic/apyrimidinic sites. Annu. Rev. Genet. 20, 201–230.
Doetsch, P. W. and Cunningham, R. P. (1990) The enzymology of apurinic/apyrimidinic endonucleases. Mutat. Res. 236, 173–201.
Demple, B. and Harrison, L. (1994) Repair of oxidative damage to DNA: enzymology and biology. Annu. Rev. Biochem. 63, 915–948.
Demple, B., Herman, T., and Chen, D. S. (1991) Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes. Proc. Natl. Acad. Sci. USA 88, 11,450–11,454.
Dizdaroglu, M., Laval, J., and Boiteux, S. (1993) Substrate specificity of the Escherichia coli endonuclease III: excision of thymine-and cytosine-derived lesions in DNA produced by radiation-generated free radicals. Biochemistry 32, 12,105–12,111.
Kow, Y. W. and Wallace, S. S. (1987) Mechanism of action of Escherichia coli endonuclease III. Biochemistry 26, 8200–8206.
Dodson, M. L., Michaels, M., and Lloyd, R. S. (1994) Unified catalytic mechanism for DNA glycosylases. J. Biol. Chem. 269, 32,709–32,712.
Bailly, V., Verly, W. G., O’Conner, T., and Laval, J. (1989) Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidine] DNA glycosylase. Biochem. J. 262, 581–589.
Yacoub, A., Augeri, L., Kelley, M. R., Doetsch, P. W., and Deutsch, W. A. (1996) A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities. EMBO J. 15, 2306–2312.
Spiering, A. L. and Deutsch, W. A. (1986) Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity. J. Biol. Chem. 261, 3222–3228.
Warner, H. R., Demple, B. F., Deutsch, W. A., Kane, C. M., and Linn, S. (1980) Apurinic/apyrimidinic endonucleases in repair of pyrimidine dimers and other lesions in DNA. Proc. Natl. Acad. Sci. USA 77, 4602–4206.
Yacoub, A., Kelley, M. R., and Deutsch, W. A. (1996) Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity. Nucleic Acids Res. 24, 4298–4303.
Deutsch, W. A. and Yacoub, A. (1999) Characterization of DNA strand cleavage by enzymes that act at abasic sites in DNA. Methods Mol. Biol. 113, 281–288.
Lindahl, T. (1980) Uracil-DNA glycosylase from Escherichia coli. Methods Enzymol. 65, 284–295.
Wilson, D. M. 3rd, Takeshita, M., Grollman, A. P., and Demple, B. (1995) Incision activity of human apurinic endonuclease (Ape) at abasic site analogs in DNA. J. Biol. Chem. 270, 16,002–16,007.
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Deutsch, W.A., Hegde, V. (2005). Analysis of DNA Strand Cleavage at Abasic Sites. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:039
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DOI: https://doi.org/10.1385/1-59259-840-4:039
Publisher Name: Humana Press
Print ISBN: 978-1-58829-084-7
Online ISBN: 978-1-59259-840-3
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