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Nucleic Acids Res. 1998 June 1; 26(11): 2771–2778.
PMCID: PMC147600

Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.


Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.

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Selected References

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  • Krokan HE, Standal R, Slupphaug G. DNA glycosylases in the base excision repair of DNA. Biochem J. 1997 Jul 1;325(Pt 1):1–16. [PubMed]
  • Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. [PubMed]
  • Loeb LA, Preston BD. Mutagenesis by apurinic/apyrimidinic sites. Annu Rev Genet. 1986;20:201–230. [PubMed]
  • Demple B, Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. [PubMed]
  • Xanthoudakis S, Smeyne RJ, Wallace JD, Curran T. The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8919–8923. [PubMed]
  • Demple B, Herman T, Chen DS. 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 U S A. 1991 Dec 15;88(24):11450–11454. [PubMed]
  • Robson CN, Hochhauser D, Craig R, Rack K, Buckle VJ, Hickson ID. Structure of the human DNA repair gene HAP1 and its localisation to chromosome 14q 11.2-12. Nucleic Acids Res. 1992 Sep 11;20(17):4417–4421. [PMC free article] [PubMed]
  • Seki S, Hatsushika M, Watanabe S, Akiyama K, Nagao K, Tsutsui K. cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III. Biochim Biophys Acta. 1992 Jul 15;1131(3):287–299. [PubMed]
  • Xanthoudakis S, Miao G, Wang F, Pan YC, Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 1992 Sep;11(9):3323–3335. [PubMed]
  • Matsumoto Y, Kim K. Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair. Science. 1995 Aug 4;269(5224):699–702. [PubMed]
  • Singhal RK, Prasad R, Wilson SH. DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. J Biol Chem. 1995 Jan 13;270(2):949–957. [PubMed]
  • Matsumoto Y, Kim K, Bogenhagen DF. Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol Cell Biol. 1994 Sep;14(9):6187–6197. [PMC free article] [PubMed]
  • Frosina G, Fortini P, Rossi O, Carrozzino F, Raspaglio G, Cox LS, Lane DP, Abbondandolo A, Dogliotti E. Two pathways for base excision repair in mammalian cells. J Biol Chem. 1996 Apr 19;271(16):9573–9578. [PubMed]
  • Kubota Y, Nash RA, Klungland A, Schär P, Barnes DE, Lindahl T. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J. 1996 Dec 2;15(23):6662–6670. [PubMed]
  • Klungland A, Lindahl T. Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. 1997 Jun 2;16(11):3341–3348. [PubMed]
  • Wilson DM, 3rd, Thompson LH. Life without DNA repair. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12754–12757. [PubMed]
  • Prasad R, Singhal RK, Srivastava DK, Molina JT, Tomkinson AE, Wilson SH. Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis. J Biol Chem. 1996 Jul 5;271(27):16000–16007. [PubMed]
  • Caldecott KW, McKeown CK, Tucker JD, Ljungquist S, Thompson LH. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol Cell Biol. 1994 Jan;14(1):68–76. [PMC free article] [PubMed]
  • Mol CD, Kuo CF, Thayer MM, Cunningham RP, Tainer JA. Structure and function of the multifunctional DNA-repair enzyme exonuclease III. Nature. 1995 Mar 23;374(6520):381–386. [PubMed]
  • Barzilay G, Mol CD, Robson CN, Walker LJ, Cunningham RP, Tainer JA, Hickson ID. Identification of critical active-site residues in the multifunctional human DNA repair enzyme HAP1. Nat Struct Biol. 1995 Jul;2(7):561–568. [PubMed]
  • Takeshita M, Chang CN, Johnson F, Will S, Grollman AP. Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. J Biol Chem. 1987 Jul 25;262(21):10171–10179. [PubMed]
  • Takeuchi M, Lillis R, Demple B, Takeshita M. Interactions of Escherichia coli endonuclease IV and exonuclease III with abasic sites in DNA. J Biol Chem. 1994 Aug 26;269(34):21907–21914. [PubMed]
  • Wilson DM, 3rd, Takeshita M, Grollman AP, Demple B. Incision activity of human apurinic endonuclease (Ape) at abasic site analogs in DNA. J Biol Chem. 1995 Jul 7;270(27):16002–16007. [PubMed]
  • Wilson DM, 3rd, Takeshita M, Demple B. Abasic site binding by the human apurinic endonuclease, Ape, and determination of the DNA contact sites. Nucleic Acids Res. 1997 Mar 1;25(5):933–939. [PMC free article] [PubMed]
  • Suh D, Wilson DM, 3rd, Povirk LF. 3'-phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends. Nucleic Acids Res. 1997 Jun 15;25(12):2495–2500. [PMC free article] [PubMed]
  • Demple B, Harrison L, Wilson DM, 3rd, Bennett RA, Takagi T, Ascione AG. Regulation of eukaryotic abasic endonucleases and their role in genetic stability. Environ Health Perspect. 1997 Jun;105 (Suppl 4):931–934. [PMC free article] [PubMed]
  • Häring M, Rüdiger H, Demple B, Boiteux S, Epe B. Recognition of oxidized abasic sites by repair endonucleases. Nucleic Acids Res. 1994 Jun 11;22(11):2010–2015. [PMC free article] [PubMed]
  • Ide H, Akamatsu K, Kimura Y, Michiue K, Makino K, Asaeda A, Takamori Y, Kubo K. Synthesis and damage specificity of a novel probe for the detection of abasic sites in DNA. Biochemistry. 1993 Aug 17;32(32):8276–8283. [PubMed]
  • Gorman MA, Morera S, Rothwell DG, de La Fortelle E, Mol CD, Tainer JA, Hickson ID, Freemont PS. The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites. EMBO J. 1997 Nov 3;16(21):6548–6558. [PubMed]
  • Shida T, Noda M, Sekiguchi J. Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI). Nucleic Acids Res. 1996 Nov 15;24(22):4572–4576. [PMC free article] [PubMed]
  • Levin JD, Johnson AW, Demple B. Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J Biol Chem. 1988 Jun 15;263(17):8066–8071. [PubMed]
  • Colvin ME, Seidl ET, Nielsen IM, Le Bui L, Hatch FT. Deprotonation and hydride shifts in nitrenium and iminium forms of aminoimidazole-azaarene mutagens. Chem Biol Interact. 1997 Dec 12;108(1-2):39–66. [PubMed]
  • Schärer OD, Nash HM, Jiricny J, Laval J, Verdine GL. Specific binding of a designed pyrrolidine abasic site analog to multiple DNA glycosylases. J Biol Chem. 1998 Apr 10;273(15):8592–8597. [PubMed]
  • McCullough AK, Dodson ML, Schärer OD, Lloyd RS. The role of base flipping in damage recognition and catalysis by T4 endonuclease V. J Biol Chem. 1997 Oct 24;272(43):27210–27217. [PubMed]
  • Coppel Y, Berthet N, Coulombeau C, Coulombeau C, Garcia J, Lhomme J. Solution conformation of an abasic DNA undecamer duplex d(CGCACXCACGC) x d(GCGTGTGTGCG): the unpaired thymine stacks inside the helix. Biochemistry. 1997 Apr 22;36(16):4817–4830. [PubMed]
  • Roberts RJ. On base flipping. Cell. 1995 Jul 14;82(1):9–12. [PubMed]
  • McCullough AK, Schärer O, Verdine GL, Lloyd RS. Structural determinants for specific recognition by T4 endonuclease V. J Biol Chem. 1996 Dec 13;271(50):32147–32152. [PubMed]
  • Vassylyev DG, Morikawa K. DNA-repair enzymes. Curr Opin Struct Biol. 1997 Feb;7(1):103–109. [PubMed]
  • Weiss B. Endonuclease II of Escherichia coli is exonuclease III. J Biol Chem. 1976 Apr 10;251(7):1896–1901. [PubMed]
  • Singer B, Hang B. What structural features determine repair enzyme specificity and mechanism in chemically modified DNA? Chem Res Toxicol. 1997 Jul;10(7):713–732. [PubMed]
  • Woodson SA, Crothers DM. Structural model for an oligonucleotide containing a bulged guanosine by NMR and energy minimization. Biochemistry. 1988 May 3;27(9):3130–3141. [PubMed]
  • Withka JM, Wilde JA, Bolton PH, Mazumder A, Gerlt JA. Characterization of conformational features of DNA heteroduplexes containing aldehydic abasic sites. Biochemistry. 1991 Oct 15;30(41):9931–9940. [PubMed]
  • Patel DJ, Kozlowski SA, Marky LA, Rice JA, Broka C, Itakura K, Breslauer KJ. Extra adenosine stacks into the self-complementary d(CGCAGAATTCGCG) duplex in solution. Biochemistry. 1982 Feb 2;21(3):445–451. [PubMed]
  • Kalnik MW, Norman DG, Swann PF, Patel DJ. Conformation of adenosine bulge-containing deoxytridecanucleotide duplexes in solution. Extra adenosine stacks into duplex independent of flanking sequence and temperature. J Biol Chem. 1989 Mar 5;264(7):3702–3712. [PubMed]
  • Kalnik MW, Norman DG, Zagorski MG, Swann PF, Patel DJ. Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution. Biochemistry. 1989 Jan 10;28(1):294–303. [PubMed]
  • Kalnik MW, Norman DG, Li BF, Swann PF, Patel DJ. Conformational transitions in thymidine bulge-containing deoxytridecanucleotide duplexes. Role of flanking sequence and temperature in modulating the equilibrium between looped out and stacked thymidine bulge states. J Biol Chem. 1990 Jan 15;265(2):636–647. [PubMed]
  • van den Hoogen YT, van Beuzekom AA, van den Elst H, van der Marel GA, van Boom JH, Altona C. Extra thymidine stacks into the d(CTGGTGCGG).d(CCGCCCAG) duplex. An NMR and model-building study. Nucleic Acids Res. 1988 Apr 11;16(7):2971–2986. [PMC free article] [PubMed]
  • van den Hoogen YT, van Beuzekom AA, de Vroom E, van der Marel GA, van Boom JH, Altona C. Bulge-out structures in the single-stranded trimer AUA and in the duplex (CUGGUGCGG).(CCGCCCAG). A model-building and NMR study. Nucleic Acids Res. 1988 Jun 10;16(11):5013–5030. [PMC free article] [PubMed]
  • Cuniasse P, Fazakerley GV, Guschlbauer W, Kaplan BE, Sowers LC. The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site. J Mol Biol. 1990 May 20;213(2):303–314. [PubMed]
  • Goljer I, Kumar S, Bolton PH. Refined solution structure of a DNA heteroduplex containing an aldehydic abasic site. J Biol Chem. 1995 Sep 29;270(39):22980–22987. [PubMed]
  • Behmoaras T, Toulme JJ, Helene C. Specific recognition of apurinic sites in DNA by a tryptophan-containing peptide. Proc Natl Acad Sci U S A. 1981 Feb;78(2):926–930. [PubMed]
  • Behmoaras T, Toulmé JJ, Hélène C. A tryptophan-containing peptide recognizes and cleaves DNA at apurinic sites. Nature. 1981 Aug 27;292(5826):858–859. [PubMed]

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