Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Aug 15;25(16):3332–3338. doi: 10.1093/nar/25.16.3332

Functional analysis of point mutations in human flap endonuclease-1 active site.

B Shen 1, J P Nolan 1, L A Sklar 1, M S Park 1
PMCID: PMC146887  PMID: 9241249

Abstract

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.

Full Text

The Full Text of this article is available as a PDF (201.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blackwell J. R., Horgan R. A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 1991 Dec 16;295(1-3):10–12. doi: 10.1016/0014-5793(91)81372-f. [DOI] [PubMed] [Google Scholar]
  2. Carr A. M., Sheldrick K. S., Murray J. M., al-Harithy R., Watts F. Z., Lehmann A. R. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene. Nucleic Acids Res. 1993 Mar 25;21(6):1345–1349. doi: 10.1093/nar/21.6.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ceska T. A., Sayers J. R., Stier G., Suck D. A helical arch allowing single-stranded DNA to thread through T5 5'-exonuclease. Nature. 1996 Jul 4;382(6586):90–93. doi: 10.1038/382090a0. [DOI] [PubMed] [Google Scholar]
  4. Goulian M., Richards S. H., Heard C. J., Bigsby B. M. Discontinuous DNA synthesis by purified mammalian proteins. J Biol Chem. 1990 Oct 25;265(30):18461–18471. [PubMed] [Google Scholar]
  5. Gutman P. D., Minton K. W. Conserved sites in the 5'-3' exonuclease domain of Escherichia coli DNA polymerase. Nucleic Acids Res. 1993 Sep 11;21(18):4406–4407. doi: 10.1093/nar/21.18.4406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harrington J. J., Lieber M. R. DNA structural elements required for FEN-1 binding. J Biol Chem. 1995 Mar 3;270(9):4503–4508. doi: 10.1074/jbc.270.9.4503. [DOI] [PubMed] [Google Scholar]
  7. Harrington J. J., Lieber M. R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 1994 Jun 1;8(11):1344–1355. doi: 10.1101/gad.8.11.1344. [DOI] [PubMed] [Google Scholar]
  8. Harrington J. J., Lieber M. R. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. 1994 Mar 1;13(5):1235–1246. doi: 10.1002/j.1460-2075.1994.tb06373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  10. Johnson R. E., Kovvali G. K., Prakash L., Prakash S. Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA. Science. 1995 Jul 14;269(5221):238–240. doi: 10.1126/science.7618086. [DOI] [PubMed] [Google Scholar]
  11. Kim Y., Eom S. H., Wang J., Lee D. S., Suh S. W., Steitz T. A. Crystal structure of Thermus aquaticus DNA polymerase. Nature. 1995 Aug 17;376(6541):612–616. doi: 10.1038/376612a0. [DOI] [PubMed] [Google Scholar]
  12. Lundquist R. C., Olivera B. M. Transient generation of displaced single-stranded DNA during nick translation. Cell. 1982 Nov;31(1):53–60. doi: 10.1016/0092-8674(82)90404-4. [DOI] [PubMed] [Google Scholar]
  13. Lyamichev V., Brow M. A., Dahlberg J. E. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993 May 7;260(5109):778–783. doi: 10.1126/science.7683443. [DOI] [PubMed] [Google Scholar]
  14. Mueser T. C., Nossal N. G., Hyde C. C. Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins. Cell. 1996 Jun 28;85(7):1101–1112. doi: 10.1016/s0092-8674(00)81310-0. [DOI] [PubMed] [Google Scholar]
  15. Murante R. S., Huang L., Turchi J. J., Bambara R. A. The calf 5'- to 3'-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage. J Biol Chem. 1994 Jan 14;269(2):1191–1196. [PubMed] [Google Scholar]
  16. Murante R. S., Rust L., Bambara R. A. Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage. J Biol Chem. 1995 Dec 22;270(51):30377–30383. doi: 10.1074/jbc.270.51.30377. [DOI] [PubMed] [Google Scholar]
  17. Murray J. M., Tavassoli M., al-Harithy R., Sheldrick K. S., Lehmann A. R., Carr A. M., Watts F. Z. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Mol Cell Biol. 1994 Jul;14(7):4878–4888. doi: 10.1128/mcb.14.7.4878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nolan J. P., Posner R. G., Martin J. C., Habbersett R., Sklar L. A. A rapid mix flow cytometer with subsecond kinetic resolution. Cytometry. 1995 Nov 1;21(3):223–229. doi: 10.1002/cyto.990210302. [DOI] [PubMed] [Google Scholar]
  19. Nolan J. P., Shen B., Park M. S., Sklar L. A. Kinetic analysis of human flap endonuclease-1 by flow cytometry. Biochemistry. 1996 Sep 10;35(36):11668–11676. doi: 10.1021/bi952840+. [DOI] [PubMed] [Google Scholar]
  20. Reagan M. S., Pittenger C., Siede W., Friedberg E. C. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J Bacteriol. 1995 Jan;177(2):364–371. doi: 10.1128/jb.177.2.364-371.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Robins P., Pappin D. J., Wood R. D., Lindahl T. Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I. J Biol Chem. 1994 Nov 18;269(46):28535–28538. [PubMed] [Google Scholar]
  22. Shen B., Nolan J. P., Sklar L. A., Park M. S. Essential amino acids for substrate binding and catalysis of human flap endonuclease 1. J Biol Chem. 1996 Apr 19;271(16):9173–9176. doi: 10.1074/jbc.271.16.9173. [DOI] [PubMed] [Google Scholar]
  23. Siegal G., Turchi J. J., Myers T. W., Bambara R. A. A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9377–9381. doi: 10.1073/pnas.89.20.9377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sommers C. H., Miller E. J., Dujon B., Prakash S., Prakash L. Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'- to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems. J Biol Chem. 1995 Mar 3;270(9):4193–4196. doi: 10.1074/jbc.270.9.4193. [DOI] [PubMed] [Google Scholar]
  25. Tishkoff D. X., Filosi N., Gaida G. M., Kolodner R. D. A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. Cell. 1997 Jan 24;88(2):253–263. doi: 10.1016/s0092-8674(00)81846-2. [DOI] [PubMed] [Google Scholar]
  26. Turchi J. J., Bambara R. A. Completion of mammalian lagging strand DNA replication using purified proteins. J Biol Chem. 1993 Jul 15;268(20):15136–15141. [PubMed] [Google Scholar]
  27. Turchi J. J., Huang L., Murante R. S., Kim Y., Bambara R. A. Enzymatic completion of mammalian lagging-strand DNA replication. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9803–9807. doi: 10.1073/pnas.91.21.9803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Waga S., Bauer G., Stillman B. Reconstitution of complete SV40 DNA replication with purified replication factors. J Biol Chem. 1994 Apr 8;269(14):10923–10934. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES