PROGRESS IN THE STRUCTURE DETERMINATION OF A HUMAN TOPOISOMERASE I - DNA COMPLEX. Matthew R. Redinbo¹, Lance Stewart¹, Ehmke Pohl¹, Xiayang Qiu¹, James J. Champoux³, and Wim G. J. Hol^1,2, ¹Department of Biological Structure and ²Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, ³Department of Microbiology, University of Washington, Seattle, WA 98195

Progress in the structure determination of a complex of human topoisomerase I and double-stranded DNA will be described. Topoisomerases are a class of ubiquitous enzymes that control the level of DNA supercoiling in cells and are vital for transcription, translation and recombination. Topoisomerase I breaks a single strand of duplex DNA, forms a transient protein-DNA complex via a phosphotyrosine bond, and allows relaxation of superhelical strain about the intact DNA strand. Eukaryotic and prokaryotic topoisomerase I enzymes appear to be distinct and to have unique mechanistic characteristics. Human topoisomerase I is a 765 amino acid (91 kD) enzyme made up of four domains. Human topoisomerase I has been shown to be the sole target of the anti-cancer compound camptothecin and of camptothecin derivatives, and is thus of significant medical interest. Elevated levels of topoisomerase I have been identified in several human cancer cell types. We have obtained diffraction-quality crystals of a complex of a recombinantly expressed 70 kD portion of human topoisomerase I which includes the active site and double-stranded DNA. Data to 2.8 resolution has been collected at -170 C from native crystals and from crystals containing an iodinated DNA oligonucleotide. Structure determination is in progress using the multiple isomorphous replacement method.