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Published August 2012 | Published + Supplemental Material
Journal Article Open

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1


In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Additional Information

© 2012 The Author(s). Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received March 12, 2012; Revised April 5, 2012; Accepted April 13, 2012. We would like to thank the Bambara and Campbell research groups for critical discussion during the course of this work and comments on the manuscript. Funding: The National Institutes of Health [GM024441 to R.A.B., GM068411 to J.W.G., GM098328-01A1 to L.B., GM078666 to J.L.C.]. Funding for open access charge: The National Institutes of Health [GM024441].

Attached Files

Published - Nucl._Acids_Res.-2012-Gloor-6774-86.pdf

Supplemental Material - nar-00693-d-2012-File009.pdf


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August 19, 2023
October 19, 2023