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J. Biol. Chem., Vol. 276, Issue 27, 25243-25253, July 6, 2001
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From the Laboratoire de Pharmacologie des Macromolécules
Biologiques, Institut Gustave Roussy, Villejuif, Cedex, 94805 France
Despite its small size (27.6 kDa), the
group I intron-encoded I-SceI endonuclease initiates intron
homing by recognizing and specifically cleaving a large intronless DNA
sequence. Here, we used gel shift assays and footprinting experiments
to analyze the interaction between I-SceI and its
target. I-SceI was found to bind to its substrate
in monomeric form. Footprinting using DNase I, hydroxyl radical,
phenanthroline copper complexes, UV/DH-MePyPs photosensitizer, and
base-modifying reagents revealed the asymmetric nature of the
interaction and provided a first glimpse into the architecture of the
complex. The protein interacts in the minor and major grooves and
distorts DNA at three distinct sites: one at the intron insertion site
and the other two, respectively, downstream (
Chemical Probing Shows That the Intron-encoded Endonuclease
I-SceI Distorts DNA through Binding in Monomeric Form
to Its Homing Site*
and
8, 9) and upstream
(+9, +10) from this site. The protein appears to stabilize the DNA
curved around it by bridging the minor groove on one face of the helix.
The scissile phosphates would lie on the outside of the bend, facing in
the same direction relative to the DNA helical axis, as expected for an
endonuclease that generates 3' overhangs. An internally
consistent model is proposed in which the protein would take advantage
of the concerted flexibility of the DNA sequence to induce a
synergistic binding/kinking process, resulting in the correct
positioning of the enzyme active site.
*
This work was supported by Grant PO1009 from the Center
National de la Recherche Scientifique and was performed in the
Laboratoire de Physique et Chimie Biomoléculaires at Institut
Curie and the Laboratoire de Chimie et Biochimie Pharmacologique et
Toxicologiques at the Faculté de Médecine, Rue des
Saint-Pères, Paris.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.
Supported by a fellowship from the French Ministère de
l'Enseignement Superieur et de la Recherche.
§
To whom correspondence should be addressed. Fax: 0147367470;
E-mail: aspassky@igr.fr.
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