Biochemical and biophysical analyses of recombinant forms of human topoisomerase I

Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH2-terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine, To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH2-terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH2- and COOH-terminally truncated core fragment encompassing residues 175-659, The specific activity of the full-length and Topo70 enzymes are indistinguishable from the native human Topo I purified from HeLa cells, Each protein is inhibited by camptothecin, topotecan, and 9-amino-camptothecin, but not by ATP. Activity is stimulated by Mg2+, Ba2+, Ca2+, Mn2+, spermine, and spermidine, The magnitude of the stimulatory effect of Mg2+ is inversely proportional to the salt concentration, Furthermore, at KCl concentrations of 300 mM or greater, the addition of Mg2+ is inhibitory, The effects of Mg2+ and the polycations spermine and spermidine are partially additive, an indication that the stimulatory mechanisms of the two substances are different, Activity was strongly inhibited or abolished by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. An examination of the hydrodynamic properties of full-length Topo I, Topo70, and Topo58 demonstrates that the core, linker, and COOH-terminal domains fold into a globular structure, while the NH2-terminal domain is highly extended, A comparison of the circular dichroism spectra of full-length Topo I and Topo70 demonstrates that residues 1-174 (similar to 21 kDa) of Topo I are largely if not completely unfolded, This observation is consistent with the fact that the NH2-terminal domain is dispensable for activity.