A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro

Several recent studies have shown that human topoisomerase I (htopol) can r ecognize various DNA lesions and thereby form a covalent topolsomerase I-DN A complex, which is known to be detrimental to cells. We have investigated whether htopol recognizes another htopol that is covalently trapped on a DN A substrate. For this purpose we created an artificial DNA substrate contai ning a specific topoisomerase I binding sequence, where the enzyme was trap ped in the covalently bound form. We demonstrate that, in vitro, free htopo l stimulates the formation of an additional cleavage complex immediately up stream of the covalently bound topolsomerase I. The predominant distance be tween the two cleavage sites is 13 nt. In addition we find that these two e nzymes may form direct protein-protein contacts and we propose that these m ay be mediated through the formation of a dimer by domain swapping involvin g the C-terminal and the core domains. Finally, we discuss the possibility that the double cleavage reaction may be the initial step for the removal o f the recognized cleavage complex.