Human exonuclease 1 threads 5'-flap substrates through its helical arch

Human exonuclease 1 (hEXO1) is a member of the 5'-nuclease superfamily and plays important roles in DNA repair. Alongside acting as a 5'-exonuclease on blunt, gapped, nicked and 3'-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5'-single-stranded flaps from duplex ends. How hEXO1 and related 5'-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5'-flaps has been controversial. Here we report the first functional data that implies that hEXO1 threads the 5'-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single-strands. Conjugation of bulky 5'-streptavidin that would "block" threading through the arch drastically slowed the hEXO1 reaction. In contrast, addition of streptavidin to a pre-formed hEXO1 5'-biotin flap DNA complex trapped a portion of the substrate in a highly-reactive threaded conformation. However, another fraction behaves as if "blocked" and decayed very slowly implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1-flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1, unifies the mode of operation for members of the 5'-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source.