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-aminocamptothecin, 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 (approximately 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.
Stewart
L
L
Department of Microbiology, School of Medicine, University of Washington, Seattle, 98195-7242, USA.
Ireton
G C
GC
Parker
L H
LH
Madden
K R
KR
Champoux
J J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002942:Circular Dichroism; D017124:Conserved Sequence; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D019772:Topotecan
D002413:Cations, Divalent; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D059004:Topoisomerase I Inhibitors; C080125:9-aminocamptothecin; D011189:Potassium Chloride; D019772:Topotecan; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I; D008274:Magnesium; D002166:Camptothecin