DNA Repair Enzymes

Thomas C. Evans1, Nicole M. Nichols1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.9
DOI:  10.1002/0471142727.mb0309s84
Online Posting Date:  October, 2008
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Abstract

In vivo DNA damage impacts the genetic stability of an organism; therefore, multiple pathways utilizing a large number of enzymes have evolved to repair DNA damage. This unit focuses on enzymes involved in base excision repair (BER). The BER enzymes possessing N‐glycosylase activity can find and remove a wide variety of damaged bases in a sea of normal bases. The combination of unique substrate specificity, accuracy, and robust in vitro activity of many of these enzymes has led to their use in various experimental techniques, including site‐specific DNA cleavage. The enzymes described in this unit are active on many substrates including oxidized purines and pyrimidines, alkylated bases, abasic sites, pyrimidine dimers, deaminated cytosines, and deaminated adenines. Curr. Protoc. Mol. Biol. 84:3.9.1‐3.9.12. © 2008 by John Wiley & Sons, Inc.

Keywords: DNA repair; N‐glycosylase; COMET assay; UDG; FPG; base excision repair

     
 
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Table of Contents

  • Introduction
  • Enzyme: Escherichia coli Uracil DNA Glycosylase
  • Enzyme: Archaeoglobus fulgidis Uracil DNA Glycosylase
  • Enzyme: Escherichia coli MutY
  • Enzyme: Human Alkyladenine‐DNA Glycosylase
  • Enzyme: Escherichia coli Formamidopyrimidine [fapy]‐DNA Glycosylase
  • Enzyme: Escherichia coli Endonuclease III
  • Enzyme: Eschericia Coli Endonuclease VIII
  • Enzyme: Human 8‐Oxoguanine DNA Glycosylase
  • Enzyme: T4 Pyrimidine Dimer Glycosylase
  • Enzyme: Escherichia coli Endonuclease IV
  • Enzyme: Human Apurinic/Apyrimidinic Endonuclease I
  • Enzyme: Esherichia Coli Endonuclease V
  • Multi‐Enzyme DNA Repair
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

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Literature Cited

Literature Cited
   Bang, D. and Church, G.M. 2008. Gene synthesis by circular assembly amplification. Nat. Methods 5:37‐39.
   Barzilay, G., Walker, L.J., Robson, C.N., and Hickson, I.D. 1995. Site‐directed mutagenesis of the human DNA repair enzyme HAP1: Identification of residues important for AP endonuclease and RNase H activity. Nucleic Acids Res. 23:1544‐1550.
   Bitinaite, J., Rubino, M., Varma, K.H., Schildkraut, I., Vaisvila, R., and Vaiskunaite, R. 2007. USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res. 35:1992‐2002.
   Collins, A.R., Dusinska, M., Gedik, C.M., and Stetina, R. 1996. Oxidative damage to DNA: Do we have a reliable biomarker? Environ. Health Perspect. 104:465‐469.
   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 32:12105‐12111.
   Erzberger, J.P. and Wilson, D.M, 3rd. 1999. The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: New insights from EDTA‐resistant incision of acyclic abasic site analogs and site‐directed mutagenesis. J. Mol. Biol. 290:447‐457.
   Gruskin, E.A. and Lloyd, R.S. 1986. The DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity. J. Biol. Chem. 261:9607‐9613.
   Hartwig, A., Dally, H., and Schlepegrell, R. 1996. Sensitive analysis of oxidative DNA damage in mammalian cells: Use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol. Lett. 88:85‐90.
   Hatahet, Z., Kow, Y.W., Purmal, A.A., Cunningham, R.P., and Wallace, S.S. 1994. New substrates for old enzymes. 5‐Hydroxy‐2′‐deoxycytidine and 5‐hydroxy‐2′‐deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N‐glycosylase, while 5‐hydroxy‐2′‐deoxyuridine is a substrate for uracil DNA N‐glycosylase. J. Biol. Chem. 269:18814‐18820.
   Hofreiter, M., Jaenicke, V., Serre, D., Haeseler, Av.A., and Pääbo, S. 2001. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res. 29:4793‐4799.
   Kerins, S.M., Collins, R., and McCarthy, T.V. 2003 Characterization of an endonuclease IV 3′‐5′ exonuclease activity J. Biol. Chem. 278:3048‐3054.
   Kutyavin, I.V., Milesi, D., Belousov, Y., Podyminogin, M., Vorobiev, A., Gorn, B., Lukhtanov, E.A., Vermeulen, N.M., and Mahoney, W. 2006. A novel endonuclease IV post‐PCR genotyping system. Nucleic Acids Res. 34:e128.
   Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil DNA glycosylase to control carry‐over contamination in polymerase chain reactions. Gene 93:125‐128.
   Melamede, R.J., Hatahat, Z., Kow, Y.W., Ide, H., and Wallace, S.S. 1994. Isolation and characterization of endonuclease VIII from Escherichia coli. Biochemistry 33:1255‐1264
   Michaels, M.L., Cruz, C., Grollman, A.P., and Miller, J.H. 1992. Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proc. Natl. Acad. Sci. U.S.A. 89:7022‐7025.
   Miyazaki, K. 2002. Random DNA fragmentation with endonuclease V: Application to DNA shuffling. Nucleic Acids Res. 30:e139.
   O'Brien, P.J. and Ellenberger, T. 2004. Dissecting the broad substrate specificity of human 3‐methyladenine‐DNA glycosylase. J. Biol. Chem. 279:9750‐9757.
   Pflaum, M., Will, O., Mahler, H.C., and Epe, B. 1998. DNA oxidation products determined with repair endonucleases in mammalian cells: Types, basal levels and influence of cell proliferation. Free Radic. Res. 29:585‐594.
   Shinmura, K., Kasai, H., Sasaki, A., Sugimura, H., and Yokota, J. 1997. 8‐hydroxyguanine (7,8‐dihydro‐8‐oxoguanine) DNA glycosylase and AP lyase activities of hOGG1 protein and their substrate specificity. Mutat. Res. 385:75‐82.
   Sidorenko, V.S., Nevinsky, G.A., and Zharkov, D.O. 2008. Specificity of stimulation of human 8‐oxoguanine–DNA glycosylase by AP endonuclease. Biochem. Biophys. Res. Comm. 368:175‐179.
   Singh, N.P., McCoy, M.T., Tice, R.R., and Schneider, E.L. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res. 175:184‐191.
   Tsai‐Wu, J.‐J., Liu, H.‐F., and Lu, A‐L. 1992. Escherichia coli MutY protein has both N‐glycosylase and apurinic/apyrimidinic endonuclease activities on A‐C and A‐G mispairs. Proc. Natl. Acad. Sci. U.S.A. 89:8779‐8783.
   Wilson, D.M., 3rd. 2005. Ape1 abasic endonuclease activity is regulated by magnesium and potassium concentrations and is robust on alternative DNA structures. J. Mol. Biol. 345:1003‐1014.
   Yao, M. and Kow, Y.W. 1994. Strand‐specific cleavage of mismatch‐containing DNA by deoxyinosine 3′‐endonuclease from Escherichia coli. J. Biol. Chem. 269:31390‐31396.
   Yao, M. and Kow, Y.W. 1995. Interaction of deoxyinosine 3′‐endonuclease from Escherichia coli with DNA containing deoxyinosine. J. Biol. Chem. 270:28609‐28616.
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