EUKARYOTIC TOPOISOMERASE I-MEDIATED CLEAVAGE REQUIRES BIPARTITE DNA INTERACTION - CLEAVAGE OF DNA SUBSTRATES CONTAINING STRAND INTERRUPTIONS IMPLICATES A ROLE FOR TOPOISOMERASE-I IN ILLEGITIMATE RECOMBINATION

Topoisomerase I-mediated cleavage has previously been demonstrated to require interaction of the enzyme with a DNA duplex region encompassin g the cleavage site (Svejstrup, J. Q., Christiansen, K., Andersen, A. H., Lund, R., and Westergaard, O (1990) J. Biol. Chem. 265, 12529-1253 5). The required region, designated region A, includes positions -5 th rough -1 on the noncleaved strand and positions -7 through +2 on the s cissile strand, relative to the cleavage site. Utilizing defined DNA s ubstrates in topoisomerase I cleavage assays we show that efficient cl eavage within region A requires additional interaction of the enzyme w ith 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 ad ditional duplex region, designated region B, was delimited to position s 6-11. The ability of topoisomerase I to interact separately with reg ions A and B was assayed on sets of DNA substrates containing a nested series of single-stranded branch sites. The obtained results demonstr ate that the normal reversible cleavage/religation equilibrium establi shed by topoisomerase I on continuous duplex DNA is replaced by irreve rsible 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 intramol ecular bipartite interaction mode of topoisomerase I during the cleava ge reaction is thus indicated by both the absence of enzyme-mediated d uplex stabilization and the wide tolerance for protruding strands betw een the cleavage site and region B. Since the irreversibly cleaved top oisomerase I-DNA complexes are kinetically competent to ligate added D NA fragments carrying free 5'-OH ends, the results suggest a role of t opoisomerase I in illegitimate recombination.