Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Jun 1;24(11):2036–2043. doi: 10.1093/nar/24.11.2036

Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.

X Wu 1, J Li 1, X Li 1, C L Hsieh 1, P M Burgers 1, M R Lieber 1
PMCID: PMC145902  PMID: 8668533

Abstract

In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.

Full Text

The Full Text of this article is available as a PDF (150.7 KB).

Selected References

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

  1. Bauer G. A., Burgers P. M. Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin. Biochim Biophys Acta. 1988 Dec 20;951(2-3):274–279. doi: 10.1016/0167-4781(88)90097-8. [DOI] [PubMed] [Google Scholar]
  2. Bendixen C., Gangloff S., Rothstein R. A yeast mating-selection scheme for detection of protein-protein interactions. Nucleic Acids Res. 1994 May 11;22(9):1778–1779. doi: 10.1093/nar/22.9.1778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgers P. M., Yoder B. L. ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends. J Biol Chem. 1993 Sep 25;268(27):19923–19926. [PubMed] [Google Scholar]
  4. Cozzarelli N. R., Kelly R. B., Kornberg A. Enzymic synthesis of DNA. 33. Hydrolysis of a 5'-triphosphate-terminated polynucleotide in the active center of DNA polymerase. J Mol Biol. 1969 Nov 14;45(3):513–531. doi: 10.1016/0022-2836(69)90309-x. [DOI] [PubMed] [Google Scholar]
  5. Fien K., Stillman B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol. 1992 Jan;12(1):155–163. doi: 10.1128/mcb.12.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Guzder S. N., Habraken Y., Sung P., Prakash L., Prakash S. Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH. J Biol Chem. 1995 Jun 2;270(22):12973–12976. doi: 10.1074/jbc.270.22.12973. [DOI] [PubMed] [Google Scholar]
  8. Habraken Y., Sung P., Prakash L., Prakash S. Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2. J Biol Chem. 1995 Dec 15;270(50):30194–30198. doi: 10.1074/jbc.270.50.30194. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Krishna T. S., Kong X. P., Gary S., Burgers P. M., Kuriyan J. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell. 1994 Dec 30;79(7):1233–1243. doi: 10.1016/0092-8674(94)90014-0. [DOI] [PubMed] [Google Scholar]
  15. Li R., Waga S., Hannon G. J., Beach D., Stillman B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature. 1994 Oct 6;371(6497):534–537. doi: 10.1038/371534a0. [DOI] [PubMed] [Google Scholar]
  16. Li X., Li J., Harrington J., Lieber M. R., Burgers P. M. Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. J Biol Chem. 1995 Sep 22;270(38):22109–22112. doi: 10.1074/jbc.270.38.22109. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. Nichols A. F., Sancar A. Purification of PCNA as a nucleotide excision repair protein. Nucleic Acids Res. 1992 Jul 11;20(13):2441–2446. doi: 10.1093/nar/20.10.2441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pawson T., Hunter T. Signal transduction and growth control in normal and cancer cells. Curr Opin Genet Dev. 1994 Feb;4(1):1–4. doi: 10.1016/0959-437x(94)90084-1. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Shivji K. K., Kenny M. K., Wood R. D. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 1992 Apr 17;69(2):367–374. doi: 10.1016/0092-8674(92)90416-a. [DOI] [PubMed] [Google Scholar]
  25. 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]
  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. 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]
  28. Zervos A. S., Gyuris J., Brent R. Mxi1, a protein that specifically interacts with Max to bind Myc-Max recognition sites. Cell. 1993 Jan 29;72(2):223–232. doi: 10.1016/0092-8674(93)90662-a. [DOI] [PubMed] [Google Scholar]

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

RESOURCES