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Published December 15, 2006 | public
Journal Article Open

Flap Endonuclease Disengages Dna2 Helicase/Nuclease from Okazaki Fragment Flaps


Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase {delta}. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing.

Additional Information

Copyright © 2006 by the American Society for Biochemistry and Molecular Biology. Received for publication, July 19, 2006, and in revised form, September 18, 2006. Originally published In Press as doi:10.1074/jbc.M606884200 on October 11, 2006 We are grateful to members of the Bambara laboratory for beneficial discussion and suggestions on the project and manuscript. We also thank Taro Masuda-Sasa and Piotr Polaczek of the Campbell laboratory for critical review of this manuscript. This work was supported by National Institutes of Health Grants GM024441 (to R.A.B.) and GM25508 (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.


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