DNA structural elements required for FEN-1 binding.

In eukaryotic cells, a 5'-flap DNA endonuclease and a double-stranded DNA 5'-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)'-exonuclease). This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiae RAD2, and S. cerevisiae YKL510. Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure. To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay. We find that the Fadj strand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1. When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops. Further investigation of the role of the Fadj strand using double flap structures reveals that the Fadj strand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand. These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.