Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1

J Biol Chem. 2000 Apr 7;275(14):10498-505. doi: 10.1074/jbc.275.14.10498.

Abstract

Human flap endonuclease 1 (FEN1), an essential DNA replication protein, cleaves substrates with unannealed 5'-tails. FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y substrate that exhibits upstream primer-independent cleavage. A pseudo-Y substrate with a sequence requiring an upstream primer for cleavage was not activated by PCNA, suggesting that PCNA does not compensate for substrate features that inhibit cleavage. A biotin.streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the cleavage site from solution. Kinetic analyses reveal that PCNA can lower the K(m) for FEN1 by 11-12-fold. Overall, our results indicate that after FEN1 tracks to the cleavage site, PCNA enhances FEN1 binding stability, allowing for greater cleavage efficiency.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA Primers
  • DNA Repair
  • Endodeoxyribonucleases / metabolism*
  • Flap Endonucleases
  • Humans
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • Proliferating Cell Nuclear Antigen / pharmacology*
  • Protein Conformation
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Templates, Genetic

Substances

  • DNA Primers
  • Oligodeoxyribonucleotides
  • Proliferating Cell Nuclear Antigen
  • Recombinant Proteins
  • Endodeoxyribonucleases
  • Flap Endonucleases
  • FEN1 protein, human