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Published March 27, 2009 | Published + Accepted Version
Journal Article Open

Significance of the dissociation of Dna2 by flap endonuclease 1 to Okazaki fragment processing in Saccharomyces cerevisiae


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.

Additional Information

© 2009 by the American Society for Biochemistry and Molecular Biology. Received for publication, December 8, 2008 , and in revised form, January 26, 2009. We would like to thank Drs. Sara Binz and Marc Wold for the purified RPA protein. We thank Dr. Tom Ryan and his team at Reichert as well as the Sullivan laboratory at the University of Rochester for training and assistance with the SPR equipment. In addition, we thank the Bambara and Campbell laboratories for beneficial discussion and review of the manuscript. This work was supported by National Institutes of Health (NIH) Grant GM024441 to R.A.B., with additional support from NIH GM087666 to J.L.C. J.A.S. was supported by NIH Grant T32 GM068411 and an Elon Huntington Hooker Graduate Fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Published - STEjbc09.pdf

Accepted Version - STEjbc09pip.pdf


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