Effect of flap modifications on human FEN1 cleavage

The flap endonuclease, FEN1, plays a critical role in DNA replication and r epair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-s pecific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism t o cleave at the point of annealing, releasing the S'-tail intact. FEN1 appe ars to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap ha ve been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could rea dily traverse this site. The footprint of the nuclease at the time of cleav age does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide br anches attached to the flap beyond the footprinted region do not prevent cl eavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-mo dified branch structures are completely inert to cleavage. These results sh ow that some flap modifications can prevent or inhibit tracking, but the tr acking mechanism tolerates a variety of flap modifications. FEN1 has a flex ible loop structure through which the flap has been proposed to thread. How ever, efficient cleavage of branched structures is inconsistent with thread ing the flap through a hole in the protein.