Reconstitution of human topoisomerase I by fragment complementation.
Human topoisomerase I (topo I, 91 kDa) is composed of four major domains; the unconserved and highly charged "N-terminal" domain (24 kDa), the conserved "core" domain (54 kDa), a poorly conserved and positively charged "linker" region (5 kDa), and the highly conserved "C-terminal" domain (8 kDa) which contains the active site tyrosine at position 723. Here we demonstrate that human topo I activity can be reconstituted by mixing a 58 kDa recombinant core domain (residues Lys175 to Ala659) with any one of a series of recombinant C-terminal fragments that range in size from 12 kDa (linker and C-terminal domains, residues Leu658 to Phe765) to 6.3 kDa (C-terminal domain residues Gln713 to Phe765). The C-terminal fragments bind tightly to the core domain, forming a 1:1 complex that is stable irrespective of ionic strength (0.01 to 1 M). The reconstituted enzymes are active only over a relatively narrow range of salt concentrations (25 to 200 mM KCl) as compared to the intact topo70 enzyme (missing the N-terminal domain). Under physiological conditions (150 mM KCl and 10 mM Mg2+) they are much more distributive in their mode of action than topo70. The reconstituted enzyme binds DNA with an affinity that is approximately 20-fold lower than that of the intact topo70. In addition, the cleavage/religation equilibrium of the reconstituted enzyme appears to be biased towards religation relative to that of the intact enzyme. Despite differences in the cleavage/religation equilibrium and affinity for DNA, the reconstituted and intact enzymes have identical sequence specificities for the cleavage of duplex DNA or suicide cleavage of oligonucleotide substrates.
Stewart
L
L
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195-7242, USA.
Ireton
G C
GC
Champoux
J J
JJ
D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007553:Isotope Labeling; D008274:Magnesium / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D017384:Sequence Deletion; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012492:Salts; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D013381:Subtilisins; D004264:DNA Topoisomerases, Type I; D008274:Magnesium