Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

DNA apurinic-apyrimidinic site binding and excision by endonuclease IV

Nature Structural & Molecular Biology volume 15, pages 515–522 (2008)Cite this article

Abstract

Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three–metal ion mechanism for damage binding and incision. The 1.10-Å resolution DNA-free and the 2.45-Å resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-Å resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV α-β triose phosphate isomerase (TIM) barrel and APE1 four-layer α-β folds of the apurinic-apyrimidinic endonuclease families.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Wild-type and mutant Endo IV structure and activity.
Figure 2: E261Q DNA substrate–bound and −free X-ray structures.
Figure 3: DNA-product bound Y72A Endo IV with implications for AP-site sugar and phosphate flipping and active-site capping.
Figure 4: Endo IV AP-site cleavage mechanism implied from structural and mutational results.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Loeb, L.A. Apurinic sites as mutagenic intermediates. Cell 40, 483–484 (1985).

    Article  CAS  Google Scholar 

  2. Sobol, R.W. et al. Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses. J. Biol. Chem. 278, 39951–39959 (2003).

    Article  CAS  Google Scholar 

  3. Lindahl, T., Karran, P. & Wood, R.D. DNA excision repair pathways. Curr. Opin. Genet. Dev. 7, 158–169 (1997).

    Article  CAS  Google Scholar 

  4. Huffman, J.L., Sundheim, O. & Tainer, J.A. DNA base damage recognition and removal: new twists and grooves. Mutat. Res. 577, 55–76 (2005).

    Article  CAS  Google Scholar 

  5. Mol, C.D., Hosfield, D.J. & Tainer, J.A. Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3′ ends justify the means. Mutat. Res. 460, 211–229 (2000).

    Article  CAS  Google Scholar 

  6. Barzilay, G. & Hickson, I.D. Structure and function of apurinic/apyrimidinic endonucleases. Bioessays 17, 713–719 (1995).

    Article  CAS  Google Scholar 

  7. Levin, J.D., Johnson, A.W. & Demple, B. Homogeneous Escherichia coli endonuclease IV. Characterization of an enzyme that recognizes oxidative damage in DNA. J. Biol. Chem. 263, 8066–8071 (1988).

    CAS  PubMed  Google Scholar 

  8. Demple, B., Herman, T. & Chen, D.S. 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, 11450–11454 (1991).

    Article  CAS  Google Scholar 

  9. Doetsch, P.W. & Cunningham, R.P. The enzymology of apurinic/apyrimidinic endonucleases. Mutat. Res. 236, 173–201 (1990).

    Article  CAS  Google Scholar 

  10. Aravind, L., Walker, D.R. & Koonin, E.V. Conserved domains in DNA repair proteins and evolution of repair systems. Nucleic Acids Res. 27, 1223–1242 (1999).

    Article  CAS  Google Scholar 

  11. Hitomi, K., Iwai, S. & Tainer, J.A. The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and repair. DNA Repair (Amst.) 6, 410–428 (2007).

    Article  CAS  Google Scholar 

  12. Demple, B., Johnson, A. & Fung, D. Exonuclease III and endonuclease IV remove 3′ blocks from DNA synthesis primers in H2O2-damaged Escherichia coli. Proc. Natl. Acad. Sci. USA 83, 7731–7735 (1986).

    Article  CAS  Google Scholar 

  13. Kerins, S.M., Collins, R. & McCarthy, T.V. Characterization of an endonuclease IV 3′-5′ exonuclease activity. J. Biol. Chem. 278, 3048–3054 (2003).

    Article  CAS  Google Scholar 

  14. Ishchenko, A.A., Yang, X., Ramotar, D. & Saparbaev, M. The 3′→5′ exonuclease of Apn1 provides an alternative pathway to repair 7,8-dihydro-8-oxodeoxyguanosine in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 6380–6390 (2005).

    Article  CAS  Google Scholar 

  15. Ischenko, A.A. & Saparbaev, M.K. Alternative nucleotide incision repair pathway for oxidative DNA damage. Nature 415, 183–187 (2002).

    Article  Google Scholar 

  16. Ishchenko, A.A., Sanz, G., Priveventzev, C.V., Maksimenko, A.V. & Saparbaev, M. Characterization of new substrate specificities of Escherichia coli and Saccharomyces cerevisiae AP endonucleases. Nucleic Acids Res. 31, 6344–6353 (2003).

    Article  CAS  Google Scholar 

  17. Levin, J.D. & Demple, B. In vitro detection of endonuclease IV-specific dna damage formed by bleomycin in vivo. Nucleic Acids Res. 24, 885–889 (1996).

    Article  CAS  Google Scholar 

  18. Ide, H. et al. α-deoxyadenosine, a major anoxic radiolysis product of adenine in DNA, is a substrate for Escherichia coli endonuclease IV. Biochemistry 33, 7842–7847 (1994).

    Article  CAS  Google Scholar 

  19. Perry, J.J. et al. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat. Struct. Mol. Biol. 13, 414–422 (2006).

    Article  CAS  Google Scholar 

  20. Hopfner, K.P. et al. Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell 105, 473–485 (2001).

    Article  CAS  Google Scholar 

  21. Hosfield, D.J., Guan, Y., Haas, B.J., Cunningham, R.P. & Tainer, J.A. Structure of the DNA repair enzyme Endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis. Cell 98, 397–408 (1999).

    Article  CAS  Google Scholar 

  22. Volbeda, A., Lahm, A., Sakiyama, F. & Suck, D. Crystal structure of Penicillium citrinum P1 nuclease at 2.8 Å resolution. EMBO J. 10, 1607–1618 (1991).

    Article  CAS  Google Scholar 

  23. Hough, E. et al. High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus. Nature 338, 357–360 (1989).

    Article  CAS  Google Scholar 

  24. Hansen, S., Hough, E., Svensson, L.A., Wong, Y.L. & Martin, S.F. Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog. J. Mol. Biol. 234, 179–187 (1993).

    Article  CAS  Google Scholar 

  25. Antikainen, N.M., Monzigo, A.F., Franklin, C.L., Robertus, J.D. & Martin, S.F. Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base. Arch. Biochem. Biophys. 417, 81–86 (2003).

    Article  CAS  Google Scholar 

  26. Bauer-Siebenlist, B. et al. Correlation of structure and function in oligonuclear Zinc(II) model phosphatases. Inorg. Chem. 43, 4189–4202 (2004).

    Article  CAS  Google Scholar 

  27. Mol, C.D., Izumi, T., Mitra, S. & Tainer, J.A. DNA-bound structures and mutants reveal abasic DNA binding by APE1 coordinates DNA repair. Nature 403, 451–456 (2000).

    Article  CAS  Google Scholar 

  28. Castagnetto, J.M. et al. MDB: the metalloprotein database and browser at The Scripps Research Institute. Nucleic Acids Res. 30, 379–382 (2002).

    Article  CAS  Google Scholar 

  29. Daniels, D.S. et al. DNA binding and nucleotide flipping by the human DNA repair protein AGT. Nat. Struct. Mol. Biol. 11, 714–720 (2004).

    Article  CAS  Google Scholar 

  30. Ishchenko, A.A. et al. Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles. Proc. Natl. Acad. Sci. USA 103, 2564–2569 (2006).

    Article  CAS  Google Scholar 

  31. Yang, X., Tellier, P., Masson, J.Y., Vu, T. & Ramotar, D. Characterization of amino acid substitutions that severely alter the DNA repair functions of Escherichia coli endonuclease IV. Biochemistry 38, 3615–3623 (1999).

    Article  CAS  Google Scholar 

  32. McMurray, C.T. & Tainer, J.A. Cancer, cadmium and genome integrity. Nat. Genet. 34, 239–241 (2003).

    Article  CAS  Google Scholar 

  33. Levin, J.D., Shapiro, R. & Demple, B. Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1. J. Biol. Chem. 266, 22893–22898 (1991).

    CAS  PubMed  Google Scholar 

  34. Kaneda, K., Sekiguchi, J. & Shida, T. Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism. Nucleic Acids Res. 34, 1552–1563 (2006).

    Article  CAS  Google Scholar 

  35. Czerwinski, R.M., Harris, T.K., Massiah, M.A., Mildvan, A.S. & Whitman, C.P. The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonase tautomerase: kinetics and structural effects of mutations of Phe-50. Biochemistry 40, 1984–1995 (2001).

    Article  CAS  Google Scholar 

  36. Ivanov, I., Tainer, J.A. & McCammon, J.A. Unraveling the three-metal-ion mechanism of the DNA repair enzyme endonuclease IV. Proc. Natl. Acad. Sci. USA 104, 1465–1470 (2007).

    Article  CAS  Google Scholar 

  37. Alberts, I.L., Nadassy, K. & Wodak, S.J. Analysis of zinc binding sites in protein crystal structures. Protein Sci. 7, 1700–1716 (1998).

    Article  CAS  Google Scholar 

  38. Jencks, W. Strain, Distortion and conformational change. in Catalysis in Chemistry and Enzymology 850 (Unabridged Dover, New York, 1987).

    Google Scholar 

  39. Parikh, S.S. et al. UDG:DNA uncleaved substrate structure is strained to enforce catalysis by coupled stereoelectronic effects. Proc. Natl. Acad. Sci. USA 97, 5083–5088 (2000).

    Article  CAS  Google Scholar 

  40. Paspaleva, K. et al. Crystal structure of the DNA repair enzyme ultraviolet damage endonuclease. Structure 15, 1316–1324 (2007).

    Article  CAS  Google Scholar 

  41. Gros, L., Ishchenko, A.A., Ide, H., Elder, R.H. & Saparbaev, M. The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway. Nucleic Acids Res. 32, 73–81 (2004).

    Article  CAS  Google Scholar 

  42. Banerjee, A., Yang, W., Karplus, M. & Verdine, G.L. Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA. Nature 434, 612–618 (2005).

    Article  CAS  Google Scholar 

  43. Wilson, S.H. & Kunkel, T.A. Passing the baton in base excision repair. Nat. Struct. Biol. 7, 866–870 (2000).

    Article  CAS  Google Scholar 

  44. Chapados, B.R. et al. Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair. Cell 116, 39–50 (2004).

    Article  CAS  Google Scholar 

  45. Hill, J.W., Hazra, T.K., Izumi, T. & Mitra, S. Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair. Nucleic Acids Res. 29, 430–438 (2001).

    Article  CAS  Google Scholar 

  46. Masuda, Y., Bennett, R.A. & Demple, B. Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product. J. Biol. Chem. 273, 30352–30359 (1998).

    Article  CAS  Google Scholar 

  47. Barik, S. Site-directed mutagenesis by double polymerase chain reaction. Mol. Biotechnol. 3, 1–7 (1995).

    Article  CAS  Google Scholar 

  48. Haas, B.J., Sandigursky, M., Tainer, J.A., Franklin, W.A. & Cunningham, R.P. Purification and characterization of Thermotoga maritima endonuclease IV, a thermostable apurinic/apyrimidinic endonuclease and 3′-repair diesterase. J. Bacteriol. 181, 2834–2839 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Ljungquist, S. A new endonuclease from Escherichia coli acting at apurinic sites in DNA. J. Biol. Chem. 252, 2808–2814 (1977).

    CAS  PubMed  Google Scholar 

  50. Cunningham, R.P. & Weiss, B. Endonuclease III (nth) mutants of Escherichia coli. Proc. Natl. Acad. Sci. USA 82, 474–478 (1985).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.A. Grasby, B.R. Chapados and R.S. Williams for insightful discussions and B. Yelent for aiding purifications. We thank the Stanford Synchrotron Radiation Laboratory, California, USA, for synchrotron facilities used in X-ray diffraction data collection. Work on DNA repair in the Tainer and Cunningham laboratories is supported by the US National Institutes of Health grants GM46312 (J.A.T. and R.P.C.) and CRR1C06RR0154464 (R.P.C.) and a Graduate Fellowship from the Skaggs Institute for Research (D.J.H.).

Author information

Author notes

  1. David J Hosfield, Brian J Haas & Magnar Björas

    Present address: Present addresses: Takeda San Diego, 10410 Science Center Drive, San Diego, California 92121, USA (D.J.H.), The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 20850, USA (B.J.H.) and Centre for Molecular Biology and Neuroscience, Institute of Medical Microbiology, Rikshospitalet University Hospital, NO-0027 Oslo, Norway (M.B.).,

Authors and Affiliations

  1. Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB4 La Jolla, 92037, California, USA

    Elsa D Garcin, David J Hosfield, Magnar Björas & John A Tainer

  2. Department of Biological Sciences, State University of New York at Albany, 1400 Washington Ave, Albany, 12222, New York, USA

    Sunil A Desai, Brian J Haas & Richard P Cunningham

Authors
  1. Elsa D Garcin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David J Hosfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sunil A Desai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brian J Haas
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Magnar Björas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Richard P Cunningham
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John A Tainer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.D.G., M.B., R.P.C. and J.A.T. analyzed data and wrote the manuscript. D.J.H. designed and performed experiments, analyzed data and wrote the manuscript. S.A.D. and B.J.H. designed and performed experiments.

Corresponding author

Correspondence to John A Tainer.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–4 and Supplementary Methods (PDF 2053 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Garcin, E., Hosfield, D., Desai, S. et al. DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. Nat Struct Mol Biol 15, 515–522 (2008). https://doi.org/10.1038/nsmb.1414

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1414

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing