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. 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.

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Selected References

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  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]

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