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J. Biol. Chem., Vol. 281, Issue 7, 3889-3898, February 17, 2006

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Nucleotide Sequence and DNA Secondary Structure, as Well as Replication Protein A, Modulate the Single-stranded Abasic Endonuclease Activity of APE1^*

Jinshui Fan, Yoshihiro Matsumoto, and David M. Wilson, III¹

From the Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224 and the Division of Medical Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

A major role of the multifunctional human Ape1 protein is to incise at apurinic/apyrimidinic (AP) sites in DNA via site-specific endonuclease activity. This nuclease function has been well characterized on double-stranded (ds) DNA substrates, where the complementary strand provides a template for subsequent base excision repair events. Recently, Ape1 was found to incise efficiently at AP sites positioned within the single-stranded (ss) regions of various biologically relevant DNA configurations. The studies within indicated that the ss endonuclease activity of Ape1 is poorly active on ss AP site-containing polyadenine or polythymine oligonucleotides, suggesting a requirement for some form of DNA secondary structure for efficient cleavage. Computational, footprinting, and biochemical analyses indicated that the nature of the secondary structure and the proximity of the AP site influence Ape1 incision efficiency significantly. Replication protein A (RPA), the major ssDNA-binding protein in mammalian cells, was found to bind ss AP-DNA with similar affinity as unmodified ssDNA and ds AP-DNA with lower affinity. Consistent with their known relative DNA binding affinities, RPA blocks/inhibits the ss, but not ds, AP endonuclease function of Ape1. Moreover, RPA inactivates Ape1 incision activity at an AP site within the ss region of a fork duplex, but not a transcription-like bubble intermediate. The data herein suggested a model whereby RPA selectively suppresses the nontemplated ss cleavage activity of Ape1 in vivo, particularly at sites of ongoing replication/recombination, by coating the ssDNA.

Received for publication, October 7, 2005 , and in revised form, December 13, 2005.

^* This work was supported by the Intramural Research Program of the NIA, National Institutes of Health. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Laboratory of Molecular Gerontology, NIA, National Institutes of Health, GRC, 2D-13, 5600 Nathan Shock Dr., Baltimore, MD. Tel.: 410-558-8153; Fax: 410-558-8157; E-mail: wilsonda{at}grc.nia.nih.gov.

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