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 c erevisiae RAD2, and S. cerevisiae YKL510. Like FEN-1, these related st ructure-specific nucleases recognize and cleave a branched DNA structu re called a DNA flap and its derivative, called a pseudo Y-structure, To dissect the important structural components of the DNA flap structu re, we have developed a mobility shift assay, We find that the F-adj s trand (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 o f the flap strand, binding efficiency drops, Further investigation of the role of the F-adj strand using double flap structures reveals that the F-adj strand is necessary to provide a double-stranded template u pon which FEN-1 can bind near the elbow of the flap strand, These resu lts provide a basis for understanding how this structure-specific nucl ease recognizes a variety of DNA substrates.