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J. Biol. Chem., Vol. 278, Issue 16, 13728-13739, April 18, 2003
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From the Department of Biochemistry and Biophysics, University of
Rochester School of Medicine and Dentistry,
Rochester, New York 14642
Flap endonuclease 1 (FEN1), involved in the
joining of Okazaki fragments, has been proposed to restrain DNA repeat
sequence expansion, a process associated with aging and disease. Here
we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we
previously found that DNA ligase I promotes expansion, but FEN1
prevents the ligation that forms expanded products. Here we show that
among the intermediates that could generate sequence expansion, a
bubble is necessary for ligation to produce the expansion product.
Severe exonuclease defects in the mutant FEN1 suggested that the
inability to degrade bubbles exonucleolytically leads to expansion.
However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we
propose that FEN1 suppresses sequence expansion by degrading flaps that
equilibrate with bubbles, thereby reducing bubble concentration. In
this way FEN1 employs endonuclease rather than exonuclease to prevent
expansions. A model is presented describing the roles of DNA structure,
DNA ligase I, and FEN1 in sequence expansion.
Analysis of Human Flap Endonuclease 1 Mutants Reveals a Mechanism
to Prevent Triplet Repeat Expansion*
*
This work was supported by National Institutes of Health
Grant GM24441.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.: 585-275-3269; Fax: 585-271-2683; E-mail: robert_bambara@urmc.rochester.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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