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J. Biol. Chem., Vol. 279, Issue 4, 2984-2992, January 23, 2004

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The Role of Lysine 532 in the Catalytic Mechanism of Human Topoisomerase I^*

Heidrun Interthal, Paulene M. Quigley, Wim G. J. Hol, and James J. Champoux¶

From the Department of Microbiology and Department of Biochemistry, University of Washington School of Medicine, Seattle, Washington 98195-7242

Based on co-crystal structures of human topoisomerase I with bound DNA, Lys⁵³² makes a minor groove contact with the strongly preferred thymidine residue at the site of covalent attachment (-1 position). Replacement of Lys⁵³² with either arginine or alanine has essentially no effect on the sequence preference of the enzyme, indicating that this interaction is not required for the preference for a T at the -1 position. Although both the cleavage and religation activities of the K532R mutant enzyme are reduced, cleavage is reduced to a greater extent than religation. The reverse is true for the K532A mutant enzyme with religation so impaired that the nicked intermediate accumulates during plasmid relaxation assays. Consistent with the shift in the cleavage religation equilibrium toward cleavage for the K532A mutant enzyme, expression of the mutant enzyme in Saccharomyces cerevisiae is cytotoxic, and thus this mutant enzyme mimics the effects of the anticancer drug camptothecin. Cleavage assays with the mutant enzymes using an oligonucleotide containing a 5'-bridging phosphorothiolate indicate that Lys⁵³² functions as a general acid during cleavage to protonate the leaving 5'-oxygen. It is possible that the contact with the -1 base is important during catalysis to provide positional rigidity to the active site. The corresponding residues in the vaccinia virus topoisomerase and the tyrosine recombinases may have similar critical roles in catalysis.

Received for publication, September 8, 2003 , and in revised form, October 30, 2003.

The atomic coordinates and structure factors (code 1R49) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Grant GM49156 from the 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.

^¶ To whom correspondence should be addressed: Dept. of Microbiology, Box 357242, University of Washington, Seattle, WA 98195-7242. Tel.: 206-543-8574; Fax: 206-543-8297; E-mail: champoux{at}u.washington.edu.

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