Okazaki fragments are initiated by short RNA/DNA primers, which are displaced into flap intermediates for processing. Flap endonuclease 1 (FEN1) and Dna2 are responsible for flap cleavage. Replication protein A (RPA)-bound flaps inhibit cleavage by FEN1 but stimulate Dna2, requiring that Dna2 cleaves prior to FEN1. Upon cleavage, Dna2 leaves a short flap, which is then cut by FEN1 forming a nick for ligation. Both enzymes require a flap with a free 5′-end for tracking to the cleavage sites. Previously, we demonstrated that FEN1 disengages the tracking mechanism of Dna2 to remove it from the flap. To determine why the disengagement mechanism evolved, we measured FEN1 dissociation of Dna2 on short RNA and DNA flaps, which occur during flap processing. Dna2 tracked onto these flaps but could not cleave, presenting a block to FEN1 entry. However, FEN1 disengaged these nonproductively bound Dna2 molecules, proceeding on to conduct proper cleavage. These results clarify the importance of disengagement. Additional results showed that flap substrate recognition and tracking by FEN1, as occur during fragment processing, are required for effective displacement of the flap-bound Dna2. Dna2 was recently shown to dissociate flap-bound RPA, independent of cleavage. Using a nuclease-defective Dna2 mutant, we reconstituted the sequential dissociation reactions in the proposed RPA/Dna2/FEN1 pathway showing that, even without cutting, Dna2 enables FEN1 to cleave RPA-coated flaps. In summary, RPA, Dna2, and FEN1 have evolved highly coordinated binding properties enabling one protein to succeed the next for proper and efficient Okazaki flap processing
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