THE CHARACTERIZATION OF A MAMMALIAN DNA STRUCTURE-SPECIFIC ENDONUCLEASE

The repair of some types of DNA double-strand breaks is thought to pro ceed through DNA flap structure intermediates. A DNA flap is a bifurca ted structure composed of double-stranded DNA and a displaced single-s trand. To identify DNA flap cleaving activities in mammalian nuclear e xtracts, we created an assay utilizing a synthetic DPI(IA flap substra te. This assay has allowed the first purification of a mammalian DNA s tructure-specific nuclease. The enzyme described here, flap endonuclea se-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single -stranded end. As expected for an enzyme which functions in double-str and break repair flap resolution, FEN-1 cleavage is flap strand-specif ic and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrate s missing one strand are not cleaved by FEN-1. Other branch structures , including Holliday junctions, are also not cleaved by FEN-1. In addi tion to endonuclease activity, FEN-1 has a 5' -3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, r ecombination and repair.