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Originally published In Press as doi:10.1074/jbc.M201765200 on April 10, 2002

J. Biol. Chem., Vol. 277, Issue 25, 22361-22369, June 21, 2002
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DNA Ligase I Competes with FEN1 to Expand Repetitive DNA Sequences in Vitro*

Leigh A. HenricksenDagger §, Janaki VeeraraghavanDagger , David R. Chafin, and Robert A. Bambara||

From the Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl2 disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.

* This work was supported by National Institutes of Health Grant GM24441 (to R. A. B.), in part by National Institutes of Health Grant GM52426 (to J. J. Hayes), and by American Cancer Society Grant RPG-00-080-01-GMC (to D. R. C.).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.

Dagger Both authors contributed equally to this work.

§ Present address: University of Rochester Medical Center, Center for Aging and Developmental Biology, 601 Elmwood Ave., Box 645, Rochester, NY 14642.

Present address: Integrated Nano-Technologies, LLC, P. O. Box 23447, Rochester, NY 14692.

|| To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-3269; Fax: 585-271-2683; E-mail: robert_bambara@urmc.rochester.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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