Bipartite Interaction Research Articles

Characterization of intra- and intermolecular DNA ligation mediated by eukaryotic topoisomerase I. Role of bipartite DNA interaction in the ligation process.

The capacity of eukaryotic topoisomerase I to catalyze intra- and intermolecular DNA strand transfer via a two-step cleavage/ligation reaction was investigated by use of purified enzyme and defined DNA substrates. Topoisomerase I-mediated cleavage requires separate interaction with a duplex region encompassing the cleavage site (region A) and a duplex region located on the side holding the 5'-OH end generated by cleavage (region B). Cleaved topoisomerase I-DNA complexes containing enzyme covalently attached at internal and terminal positions were employed to characterize the intra- and intermolecular ligation reactions. Enzyme attached covalently at an internal position of a partially single-stranded DNA molecule is able to catalyze ligation of a complementary dinucleotide within region A in the absence of interaction with region B. Moreover, the dinucleotide confines the minimal DNA acceptor for intramolecular ligation. Topoisomerase I attached covalently to DNA at a terminal position can ligate the cleaved strand to heterologous duplex DNA regardless of sequence, whereas ligation does not proceed with single-stranded DNA. When these features are considered together with the observation that intermolecular ligation is inhibited by 1 M NaCl, it suggests that the reaction requires bipartite DNA interaction. A model is proposed that relates the bipartite DNA binding of eukaryotic topoisomerase I to the catalytic functions.

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 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.