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J. Biol. Chem., Vol. 275, Issue 32, 24630-24638, August 11, 2000

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Assignment of Functional Amino Acids around the Active Site of Human DNA Topoisomerase II^*

Yoshito Okada, Yasutomo Ito^§, Akihiko Kikuchi^¶, Yuji Nimura, Shonen Yoshida^§, and Motoshi Suzuki^§

From the  First Department of Surgery and the Laboratories of ^§ Cancer Cell Biology and ^¶ Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, 466-8550, Japan

An expression library for active site mutants of human topoisomerase II (TOP2) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2 mutants, no substitution was allowed at Tyr⁸⁰⁵, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu⁷⁹⁴, Asp⁷⁹⁷, Ala⁸⁰¹, and Arg⁸⁰⁴. Thus, these 5 residues are critical for human TOP2 activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser⁸⁰³ and Lys⁷⁹⁸, which confer drug resistance, lie adjacent to this plane.

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