STRUCTURAL STUDIES OF TYPE I DNA TOPOISOMERASES Alfonso Mondragón, Christopher Lima, Neal Lue, Alexandra Patera, and Amit Sharma. Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA

DNA topoisomerases are proteins responsible for controlling and maintaining the topological state of DNA in the cell. They have been found in all cell types of both eukaryotes and prokaryotes and additionally in some viruses. They are involved in DNA replication, transcription, and genetic recombination. All topoisomerases work by forming a transient break in DNA through a phosphotyrosine bond. Type I topoisomerases break one DNA strand at a time and then pass another strand through the transient break. No external energy source is required for this reaction as the bond energy is conserved.

We have identified and crystallized a 67 kDa domain of Escherichia coli DNA topoisomerase I containing the catalytic tyrosine that is capable of cleaving single stranded DNA. The structure of the 67 kDa fragment has been determined and refined to 1.9Å resolution. The fragment shows a novel structural fold, with the amino and carboxy termini forming a large, globular domain and the central region forming a torus inside which DNA may bind. We have also solved the structure of the intact E. coli DNA topoisomerase III, a close relative of topoisomerase I. The two structures show clear structural similarities and probably share similar mechanism of action.

The structure of E. coli DNA topoisomerase I has provided a wealth of information on prokaryotic-like type I DNA topoisomerases. To further our understanding of the other major sub-family of type I topoisomerases, we have solved the structure of a 27 kDa fragment of S. cerevisiae DNA topoisomerase I and of a 9 kDa amino terminal fragment of vaccinia virus DNA topoisomerase I. The structure of the former shows a novel architecture with two domains linked by a pair of alpha helices forming an elbow and a polyproline helix. Together with new biochemical data, the structure suggests that this fragment is directly involved in interactions with DNA. The structure of the vaccinia virus fragment forms a five stranded beta-sheet with two short alpha helices. The lack of structural similarity with the S. cerevisiae structure clearly suggests that these two proteins may not be related.