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Originally published In Press as doi:10.1074/jbc.M104632200 on July 5, 2001

J. Biol. Chem., Vol. 276, Issue 36, 34213-34220, September 7, 2001
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Role of DNA End Distortion in Catalysis by Avian Sarcoma Virus Integrase*

Richard A. KatzDagger , Paul DiCandeloro§, George Kukolj, and Anna Marie Skalka

From the Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111

Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the avian sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn2+ as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.

* This work was supported by National Institutes of Health Grants AI40385, CA71515, and CA06927 (to A. M. S.) and by an appropriation from the Commonwealth of Pennsylvania (to A. M. S.).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 To whom correspondence should be addressed: Fox Chase Cancer Center, Institute for Cancer Research, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3668; Fax: 215-728-2778; E-mail: R_Katz@fccc.edu.

§ Current address: DuPont Pharmaceuticals, Experimental Station, E336/1b, Wilmington, DE 19880.

Current address: Boehringer Ingelheim (Canada) Ltd. Research and Development, Laval, Canada H7S 2G5.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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