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Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae

Stewart, Jason A. and Miller, Adam S. and Campbell, Judith L. and Bambara, Robert A. (2008) Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae. Journal of Biological Chemistry, 283 (46). pp. 31356-31365. ISSN 0021-9258. PMCID PMC2581594. doi:10.1074/jbc.M805965200.

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Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer, which is removed before the fragments are joined. Polymerase d displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Since Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1.

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Additional Information:© 2008 the American Society for Biochemistry and Molecular Biology. Submitted on August 1, 2008. Revised on September 16, 2008. Accepted on September 17, 2008. Papers In Press, published online ahead of print September 17, 2008, 10.1074/jbc.M805965200. We would like to thank Drs. Sara Binz and Marc Wold for the purified yeast and human RPA proteins. In addition, we would like to thank the Bambara laboratory for valuable discussion and Dr. Judy Campbell and her laboratory for critical review of the manuscript. This work was supported by National Institutes of Health Grant (NIH) GM024441 to R.A.B., with additional support from NIH GM087666 to J.L.C. 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. J.A.S. was supported by NIH Grant T32 GM068411 from an Institutional Ruth L. Kirschstein National Research Service Award.
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NIH Predoctoral FellowshipT32 GM068411
Issue or Number:46
PubMed Central ID:PMC2581594
Record Number:CaltechAUTHORS:STEjbc08
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11736
Deposited By: Archive Administrator
Deposited On:22 Sep 2008 17:59
Last Modified:08 Nov 2021 22:02

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