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J Biol Chem, Vol. 275, Issue 14, 10498-10505, April 7, 2000
From the Department of Biochemistry and Biophysics and Cancer
Center, University of Rochester School of Medicine and Dentistry,
Rochester, New York 14642
Human flap endonuclease 1 (FEN1), an essential
DNA replication protein, cleaves substrates with unannealed 5'-tails.
FEN1 apparently tracks along the flap from the 5'-end to the cleavage site. Proliferating cell nuclear antigen (PCNA) stimulates FEN1 cleavage 5-50-fold. To determine whether tracking, binding, or cleavage is enhanced by PCNA, we tested a variety of flap substrates. Similar levels of PCNA stimulation occur on both a cleavage-sensitive nicked substrate and a less sensitive gapped substrate. PCNA stimulates FEN1 irrespective of the flap length. Stimulation occurs on a pseudo-Y
substrate that exhibits upstream primer-independent cleavage. A
pseudo-Y substrate with a sequence requiring an upstream primer for
cleavage was not activated by PCNA, suggesting that PCNA does not
compensate for substrate features that inhibit cleavage. A biotin·streptavidin conjugation at the 5'-end of a flap structure prevents FEN1 loading. The addition of PCNA does not restore FEN1 activity. These results indicate that PCNA does not direct FEN1 to the
cleavage site from solution. Kinetic analyses reveal that PCNA can
lower the Km for FEN1 by 11-12-fold. Overall, our
results indicate that after FEN1 tracks to the cleavage site, PCNA
enhances FEN1 binding stability, allowing for greater cleavage efficiency.
Mechanism Whereby Proliferating Cell Nuclear Antigen Stimulates
Flap Endonuclease 1*
*
This work was supported by National Institutes of Health
Grant GM24441 and fellowship Grant GM18961 (to L. A. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Rochester Medical Center, Dept. of Biochemistry and Biophysics, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 716-275-3269; Fax:
716-271-2683; E-mail: robert_bambara@urmc.rochester.edu.
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