Author(s): Stewart L, Ireton GC, Champoux JJ
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Abstract 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.
This article was published in J Mol Biol
and referenced in Journal of Antivirals & Antiretrovirals