Flap endonuclease 1 mechanism analysis indicates flap base binding prior to threading

J Biol Chem. 2010 Nov 5;285(45):34922-31. doi: 10.1074/jbc.M110.165902. Epub 2010 Aug 25.

Abstract

FEN1 cleaves 5' flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5'-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5'-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5'-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Flap Endonucleases / chemistry*
  • Flap Endonucleases / genetics
  • Flap Endonucleases / metabolism
  • Protein Binding

Substances

  • Escherichia coli Proteins
  • DNA
  • Flap Endonucleases