Abstract
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.
Highlights
Zaki fragment is between 100 and 150 nucleotides3 in length
We have recently shown that the addition of Pif1 in reconstituted Okazaki fragment processing augmented the subset of longer flaps that escaped flap endonuclease 1 (FEN1) cleavage and were bound by replication protein A (RPA) [29]
The ability of FEN1 to cleave an RNA flap indicates that it is designed to act constantly on the RNA, and DNA, as the flap is generated. In support of this conclusion, biochemical reconstitution studies suggest that coordination between pol ␦ and FEN1 produces short cleavage products of 1– 8 nt, with the majority of products being mononucleotides
Summary
GCT GGC ACG GTC GGA TTT AAA GTT CGG TGG GCA GGT GGG CTG CG GCG GTC CCA AAA GGG TCA GTG CTG GGC AAA ATG TTG CTG CAC TGA CCC G GCT GGC ACG GTC GGA TTT AAA GTT AGG GCA GGT GGG CTG CGG TGG AGG ACG a Nucleotides shown in boldface type are biotinylated. b RNA segment is shown in italic type. c Underlined nucleotide indicates the last annealed nucleotide. We showed that mostly short flaps were created during strand displacement by pol ␦, a minor subset of longer flaps arose [27] This subset reached a length at which RPA could stably bind, suggesting a role for Dna in processing at least some flaps. We have recently shown that the addition of Pif in reconstituted Okazaki fragment processing augmented the subset of longer flaps that escaped FEN1 cleavage and were bound by RPA [29]. These results suggest that Pif aids pol ␦ strand displacement in creating long flap substrates that require Dna nuclease function. We tested the proposed sequential dissociation reactions by reconstituting the RPA/Dna2/FEN1 pathway with the nuclease-defective Dna E675A
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