Topoisomerase I-mediated cleavage has previously been demonstrated to require interaction of the enzyme with a DNA duplex region encompassing the cleavage site (Svejstrup, J. Q., Christiansen, K., Andersen, A. H., Lund, R., and Westergaard, O (1990) J. Biol. Chem. 265, 12529-12535). The required region, designated region A, includes positions -5 through -1 on the noncleaved strand and positions -7 through +2 on the scissile strand, relative to the cleavage site. Utilizing defined DNA substrates in topoisomerase I cleavage assays we show that efficient cleavage within region A requires additional interaction of the enzyme with duplex DNA on the side holding the 5'-OH end generated by cleavage. By analyzing the interaction of topoisomerase I with DNA substrates varying by single nucleotides on either strand outside region A, an additional duplex region, designated region B, was delimited to positions 6-11. The ability of topoisomerase I to interact separately with regions A and B was assayed on sets of DNA substrates containing a nested series of single-stranded branch sites. The obtained results demonstrate that the normal reversible cleavage/religation equilibrium established by topoisomerase I on continuous duplex DNA is replaced by irreversible cleavage on DNA substrates containing branch sites between the cleavage site and region B as these DNA substrates allow cleavage but prevent religation due to release of the incised strands. The intramolecular bipartite interaction mode of topoisomerase I during the cleavage reaction is thus indicated by both the absence of enzyme-mediated duplex stabilization and the wide tolerance for protruding strands between the cleavage site and region B. Since the irreversibly cleaved topoisomerase I-DNA complexes are kinetically competent to ligate added DNA fragments carrying free 5'-OH ends, the results suggest a role of topoisomerase I in illegitimate recombination.