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:

Roles of divalent metal ions in flap endonuclease–substrate interactions

Nature Structural & Molecular Biology volume 11, pages 450–456 (2004)Cite this article

Abstract

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.

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: Structure of T5FEN metal-binding sites.
Figure 2: Metal ion concentration modulates T5FEN activity.
Figure 3: Ca2+ ions stabilize T5FEN–DNA complexes.
Figure 4: Site II mutation causes selective loss of exonuclease activity.
Figure 5: Isothermal titration calorimetry.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Cowan, J.A. Metal activation of enzymes in nucleic acid biochemistry. Chem. Rev. 98, 1067–1088 (1998).

    Article  CAS  Google Scholar 

  2. Lieber, M.R. The FEN1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. Bioessays 19, 233–240 (1997).

    Article  CAS  Google Scholar 

  3. Ceska, T.A. & Sayers, J.R. Structure-specific DNA cleavage by 5′ nucleases. Trends Biochem. Sci. 23, 331–336 (1998).

    Article  CAS  Google Scholar 

  4. Kucherlapati, M. et al. Haploinsufficiency of flap endonuclease (Fen1) leads to rapid tumor progression. Proc. Natl. Acad. Sci. USA 99, 9924–9929 (2002).

    Article  CAS  Google Scholar 

  5. Harrington, J.J. & Lieber, M.R. The characterisation of a mammalian DNA structure-specific endonuclease. EMBO J. 13, 1235–1246 (1994).

    Article  CAS  Google Scholar 

  6. Lyamichev, V., Brow, M.A.D. & Dahlberg, J.E. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260, 778–783 (1993).

    Article  CAS  Google Scholar 

  7. Diaz, A., Pons, M.E., Lacks, S.A. & Lopez, P. Streptococcus pneumoniae DNA polymerase I lacks 3′–5′ exonuclease activity: localization of the 5′–3′ exonucleolytic domain. J. Bacteriol. 174, 2014–2024 (1992).

    Article  CAS  Google Scholar 

  8. Bhagwat, M. & Nossal, N.G. Bacteriophage T4 RNase H removes both RNA primers and adjacent DNA from the 5′ end of lagging strand fragments. J. Biol. Chem. 276, 28516–28524 (2001).

    Article  CAS  Google Scholar 

  9. Rumbaugh, J.A., Murante, R.S., Shi, S. & Bambara, R.A. Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing. J. Biol. Chem. 272, 22591–22599 (1997).

    Article  CAS  Google Scholar 

  10. Sayers, J.R. & Artymiuk, P.J. Flexible loops and helical arches. Nat. Struct. Biol. 5, 668–670 (1998).

    Article  CAS  Google Scholar 

  11. Kim, Y. et al. Crystal structure of Thermus aquaticus DNA polymerase. Nature 376, 612–616 (1995).

    Article  CAS  Google Scholar 

  12. Mueser, T.C., Nossal, N.G. & Hyde, C.C. Structure of bacteriophage T4 RNase H, a 5′–3′ RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins. Cell 85, 1101–1112 (1996).

    Article  CAS  Google Scholar 

  13. Ceska, T.A., Sayers, J.R., Stier, G. & Suck, D. A helical arch allowing single-stranded DNA to thread through T5 5′ exonuclease. Nature 382, 90–93 (1996).

    Article  CAS  Google Scholar 

  14. Hwang, K.Y., Baek, K., Kim, H.Y. & Cho, Y. The crystal structure of flap endonuclease-1 from Methanococcus jannaschii . Nat. Struct. Biol. 5, 707–713 (1998).

    Article  CAS  Google Scholar 

  15. Hosfield, D.J., Mol, C.D., Shen, B. & Tainer, J.A. Structure of the DNA repair and replication endonuclease and exonuclease FEN1: coupling DNA and PCNA binding to FEN1 activity. Cell 95, 135–146 (1998).

    Article  CAS  Google Scholar 

  16. Matsui, E., et al. Molecular structure and novel DNA binding sites located in loops of flap endonuclease-1 from Pyrococcus horikoshii . J. Biol. Chem. 277, 37840–37847 (2002).

    Article  CAS  Google Scholar 

  17. Beese, L.S. & Steitz, T.A. Structural basis for the 3′–5′ exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25–33 (1991).

    Article  CAS  Google Scholar 

  18. Bhagwat, M., Meara, D. & Nossal, N.G. Identification of residues of T4 RNase H required for catalysis and DNA binding J. Biol. Chem. 272, 28531–28538 (1997).

    Article  CAS  Google Scholar 

  19. Amblar, M. & Lopez, P. Purification and properties of the 5′–3′ exonuclease D190A mutant of DNA polymerase I from Streptococcus pneumoniae . Eur. J. Biochem. 252, 124–132 (1998).

    Article  CAS  Google Scholar 

  20. Mizrahi, V. & Huberts, P. Deoxy- and dideoxynucleotide discrimination and identification of critical 5′ nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis . Nucleic Acids Res. 24, 4845–4852 (1996).

    Article  CAS  Google Scholar 

  21. Xu, Y. et al. Biochemical and mutational studies of the 5′–3′ exonuclease of DNA polymerase I of Escherichia coli . J. Mol. Biol. 268, 284–302 (1997).

    Article  CAS  Google Scholar 

  22. Xu, Y., Potapova, O., Leschziner, A.E., Grindley, N.D. & Joyce, C.M. Contacts between the 5′ nuclease of DNA polymerase I and its DNA substrate. J. Biol. Chem. 276, 30167–30177 (2001).

    Article  CAS  Google Scholar 

  23. Shen, B., Nolan, J.P., Sklar, L.A. & Park, M.S. Functional analysis of point mutations in human flap endonuclease-1 active site. Nucleic Acids Res. 25, 3332–3338 (1997).

    Article  CAS  Google Scholar 

  24. Amblar, M., de Lacoba, M.G., Corrales, M.A. & Lopez, P. Biochemical analysis of point mutations in the 5′–3′ exonuclease of DNA polymerase I of Streptococcus pneumoniae. Functional and structural implications. J. Biol. Chem. 276, 19172–19181 (2001).

    Article  CAS  Google Scholar 

  25. Tock, M.R., Frary, E., Sayers, J.R. & Grasby, J.A. Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease. EMBO J. 22, 995–1004 (2003).

    Article  CAS  Google Scholar 

  26. Haq, I., Chowdhry, B.Z. & Jenkins, T.C. Calorimetric techniques in the study of high-order DNA-drug interactions. Methods Enzymol. 340, 109–149 (2001).

    Article  CAS  Google Scholar 

  27. Wiseman, T., Williston, S., Brandts, J.F. & Lin, L.N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989).

    Article  CAS  Google Scholar 

  28. Milos, M., Schaer, J.J., Comte, M. & Cox, J.A. Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin. J. Biol. Chem. 262, 2746–2749 (1987).

    CAS  PubMed  Google Scholar 

  29. Ogawa, Y. & Tanokura, M. Calcium binding to calmodulin: effects of ionic strength, Mg2+, pH and temperature. J. Biochem. 95, 19–28 (1984).

    Article  CAS  Google Scholar 

  30. Jose, T.J., Conlan, L.H. & Dupureur, C.M. Quantitative evaluation of metal ion binding to PvuII restriction endonuclease. J. Biol. Inor. Chem. 4, 814–823 (1999).

    Article  CAS  Google Scholar 

  31. Zheng, L., Li, M., Shan, J., Krishnamoorthi, R. & Shen, B. Distinct roles of two Mg2+ binding sites in regulation of murine flap endonuclease-1 activities. Biochemistry 41, 10323–10331 (2002).

    Article  CAS  Google Scholar 

  32. Hosfield, D.J., Frank, G., Weng, Y., Tainer, J.A. & Shen, B. Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1. J. Biol. Chem. 273, 27154–27161 (1998).

    Article  CAS  Google Scholar 

  33. Sayers, J.R. & Eckstein, F. Properties of overexpressed phage T5 D15 exonuclease. J. Biol. Chem. 265, 18311–18317 (1990).

    CAS  PubMed  Google Scholar 

  34. Vipond, I.B. & Halford, S.E. Specific DNA recognition by EcoRV restriction endonuclease induced by calcium ions. Biochemistry (US), 34, 1113–1119 (1995).

    Article  CAS  Google Scholar 

  35. Sayers, J.R. & Eckstein, F. A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease. Nucleic Acids Res. 19, 4127–4132 (1991).

    Article  CAS  Google Scholar 

  36. Garforth, S.J. & Sayers, J.R. Structure-specific DNA binding by bacteriophage T5 5′–3′ exonuclease. Nucleic Acids Res. 25, 3801–3807 (1997).

    Article  CAS  Google Scholar 

  37. Sayers, J.R. Viral polymerase-associated 5′–3′ exonucleases: expression, purification, and uses. Methods Enzymol. 275, 227–238 (1996).

    Article  CAS  Google Scholar 

  38. Fraser, M.J. Purification and properties of Neurospora crassa endo-exonuclease, an enzyme which can be converted to a single-strand specific endonuclease. Methods Enzymol. 65, 255–263 (1980).

    Article  CAS  Google Scholar 

  39. Garforth, S.J., Ceska, T.A., Suck, D. & Sayers, J.R. Mutagenesis of conserved lysine residues in bacteriophage T5 5′–3′ exonuclease suggests separate mechanisms of endo- and exonucleolytic cleavage. Proc. Natl. Acad. Sci. USA 96, 38–43 (1999).

    Article  CAS  Google Scholar 

  40. Ceska, T.A., Sayers, J.R., Eckstein, F. & Suck, D. Preliminary crystallographic studies on the D15 5′–3′ exonuclease from phage T5. J. Mol. Biol. 233, 179–182 (1993).

    Article  CAS  Google Scholar 

  41. Kabsch, W. Evaluation of single crystal X-ray diffraction data from a position sensitive detector. J. Appl. Crystallogr. 21, 916–924 (1988).

    Article  CAS  Google Scholar 

  42. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

  43. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  44. Badger, J., Kumar, R.A., Yip, P. & Szalma, S. New features and enhancements in the X-PLOR computer program. Proteins 35, 25–33 (1999).

    Article  CAS  Google Scholar 

  45. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Grasby for informal discussions and critical reading of the manuscript. This paper is dedicated to the memory of A. Jones. The support of The Wellcome Trust (grant numbers 052123 and 058034) is gratefully acknowledged. The White Rose University Consortium and the BBSRC supported studentships for M.F. and J.J.D., respectively.

Author information

Author notes

  1. Thomas Ceska

    Present address: Celltech R&D Ltd., 216 Bath Road, Slough, SL1 4EN, UK

Authors and Affiliations

  1. Division of Genomic Medicine, University of Sheffield School of Medicine and Biomedical Science, Beech Hill Road, Sheffield, S10 2RX, UK

    Min Feng, Dipak Patel, Joe J Dervan & Jon R Sayers

  2. Structural and Computational Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany

    Thomas Ceska & Dietrich Suck

  3. Department of Chemistry, Centre for Chemical Biology, University of Sheffield, Sheffield, S3 7HF, UK

    Ihtshamul Haq

Authors
  1. Min Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dipak Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joe J Dervan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Ceska
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dietrich Suck
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ihtshamul Haq
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jon R Sayers
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jon R Sayers.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Feng, M., Patel, D., Dervan, J. et al. Roles of divalent metal ions in flap endonuclease–substrate interactions. Nat Struct Mol Biol 11, 450–456 (2004). https://doi.org/10.1038/nsmb754

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb754

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