Abstract
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.
Highlights
The human topoisomerase I (Topo I) is composed of 765 residues with a predicted molecular mass of 91 kDa
Expression and Purification of Recombinant Forms of Human Topo I—With the long term goal of investigating the domain structure of human Topo I, we developed procedures to obtain large quantities of purified enzyme
This was achieved by the generation of recombinant baculoviruses that express wild type and active-site mutant (Y723F) versions of the fulllength human Topo I and a 70-kDa protein (Topo70) which is missing the first 174 NH2-terminal amino acids (Fig. 2A)
Summary
All plasmids were constructed by common subcloning techniques and propagated in either the SureTM (Stratagene) or TOP10FЈ (Invitrogen) strains of Escherichia coli. The dialyzed PEG supernatant was diluted with an equal volume of water, filtered through a 0.45-m filter, and loaded onto a POROS SP20 (4.6/100) column that was equilibrated with cation exchange buffer (7 mM MES, 7 mM HEPES, 7 mM sodium acetate, pH 7.5) plus 100 mM NaCl. The SP20 column was eluted with a 30-ml linear salt gradient from 100 to 800 mM NaCl. Plasmid relaxation assays were performed to identify the peak Topo I fractions, which were found to elute at ϳ700 mM NaCl. The Topo I fractions were pooled, dialyzed against 2 liters of PPB, and passed over a 2-ml bed volume of PS that was equilibrated with PPB. The dialyzed PEG supernatant was clarified by centrifugation at 10,000 ϫ g for 10 min and loaded onto a POROS SP20 (4.6/100) column that was equilibrated with cation exchange buffer plus 400 mM NaCl. The proteins were eluted with a linear 50-ml NaCl gradient from 400 mM to 1 M. Autoradiography was performed by exposing dried gels to Kodak XAR film
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