RECONSTITUTION OF HUMAN TOPOISOMERASE-I BY FRAGMENT COMPLEMENTATION

Human topoisomerase I (topo I, 91 kDa) is composed of four major domai ns; the unconserved and highly charged ''N-terminal'' domain (24 kDa), the conserved ''core'' domain (54 kDa), a poorly conserved and positi vely charged ''linker'' region (5 kDa), and the highly conserved ''C-t erminal'' domain (8 kDa) which contains the active site tyrosine at po sition 723. Here we demonstrate that human topo I activity can be reco nstituted by mixing a 58 kDa recombinant core domain (residues Lys175 to Ala659) with any one of a series of recombinant C-terminal fragment s that range in size from 12 kDa (linker and C-terminal domains, resid ues Leu658 to Phe765) to 6.3 kDa (C-terminal domain residues Gln713 to Phe765). The C-terminal fragments bind tightly to the core domain, fo rming a 1:1 complex that is stable irrespective of ionic strength (0.0 1 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 physi ological conditions (150 mM KCl and 10 mM Mg2+) they are much more dis tributive in their mode of action than topo70. The reconstituted enzym e binds DNA with an affinity that is similar to 20-fold lower than tha t of the intact topo70. In addition, the cleavage/religation equilibri um of the reconstituted enzyme appears to be biased towards religation relative to that of the intact enzyme. Despite differences in the cle avage/religation equilibrium and affinity for DNA, the reconstituted a nd intact enzymes have identical sequence specificities for the cleava ge of duplex DNA or suicide cleavage of oligonucleotide substrates. (C ) 1997 Academic Press Limited.