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Originally published In Press as doi:10.1074/jbc.M705781200 on December 4, 2007

J. Biol. Chem., Vol. 283, Issue 6, 3305-3315, February 8, 2008
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Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin*

Marié van der Merwe and Mary-Ann Bjornsti1

From the Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended {alpha}-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short {alpha}-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this {alpha}-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.


Received for publication, July 13, 2007 , and in revised form, November 7, 2007.

* This work was supported in part by National Institutes of Health Grants CA58755 (to M.-A. B.), NCI Cancer Center Core Grant CA21765, and the American Lebanese Syrian Associated Charities. 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.

1 To whom correspondence should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis TN 38105. Tel.: 901-495-2315; Fax: 901-495-4290; E-mail: mary-ann.bjornsti{at}stjude.org.


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