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J. Biol. Chem., Vol. 279, Issue 49, 50840-50849, December 3, 2004

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Dna2p Helicase/Nuclease Is a Tracking Protein, Like FEN1, for Flap Cleavage during Okazaki Fragment Maturation^*

Hui-I Kao, Judith L. Campbell¶, and Robert A. Bambara||

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 and the ^¶Braun Laboratories, California Institute of Technology, Pasadena, California 91125

During cellular DNA replication the lagging strand is generated as discontinuous segments called Okazaki fragments. Each contains an initiator RNA primer that is removed prior to joining of the strands. Primer removal in eukaryotes requires displacement of the primer into a flap that is cleaved off by flap endonuclease 1 (FEN1). FEN1 employs a unique tracking mechanism that requires the recognition of the free 5' terminus and then movement to the base of the flap for cleavage. Abnormally long flaps are coated by replication protein A (RPA), inhibiting FEN1 cleavage. A second nuclease, Dna2p, is needed to cleave an RPA-coated flap producing a short RPA-free flap, favored by FEN1. Here we show that Dna2p is also a tracking protein. Annealed primers or conjugated biotin-streptavidin complex block Dna2p entry and movement. Single-stranded binding protein-coated flaps inhibit Dna2p cleavage. Like FEN1, Dna2p can track over substrates with a non-Watson Crick base, such as a biotin, or a missing base within a chain. Unlike FEN1, Dna2p shows evidence of a "threading-like" mechanism that does not support tracking over a branched substrate. We propose that the two nucleases both track, Dna2p first and then FEN1, to remove initiator RNA via long flap intermediates.

Received for publication, August 12, 2004 , and in revised form, September 22, 2004.

^* This work was supported by National Institutes of Health Grants GM24441 (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.

Present address: Dept. of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-3269; Fax: 585-275-6007; E-mail: robert_bambara{at}urmc.rochester.edu.

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