RAMAN AND CD SPECTROSCOPY OF RECOMBINANT 68-KDA DNA HUMAN TOPOISOMERASE-I AND ITS COMPLEX WITH SUICIDE DNA-SUBSTRATE

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were emplo yed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oli gonucleotide (solig) binding and cleavage, Spectroscopic data combined with statistical prediction techniques were employed to construct a m odel of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structur es. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix o f 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also show s redistribution of the structural rotamers of the 68-kDa topo I disul fide bonds and significant changes in the I-I-bonding of the Tyr resid ues and in the microenvironment/conformation of the Trp side chains. N o structural modifications of the DNA substrate were detected by spect roscopic techniques. The data presented provide the first direct exper imental evidence of the human topo I conformational transition after t he cleavage step in the reaction of binding and cleavage of DNA substr ate by the enzyme. This evidence supports the model of the enzyme func tion requiring the protein conformational transition. The most probabl e location of the enzyme transformations was the core and the C-termin al conservative 68-kDa topo I structural domains. By contrast, the lin ker domain was found to have an extremely low potential for solig-indu ced structural transformations. The pattern of redistribution of prote in secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mo de of topo I/DNA interaction in the substrate binding and cleavage rea ction.