A 52 base pair alternating purine-pyrimidine (RY) repeat sequence lies in the 5' upstream region of the human beta-globin gene. The structural transition of a plasmid containing this repeat was analyzed by two-dimensional gel electrophoresis. These conformational studies indicate that the 52 bp RY repeat undergoes local transition from the right-handed B-DNA into a cruciform DNA under torsional stress and the transition initiates at a threshold level of negative supercoiling (-sigma = 0.042). The superhelicity-dependent S1 nuclease cleavage sites were mapped only within the RY repeat and no nicking was observed outside of the repeat. In view of the fact that DNA topoisomerase II is highly reactive towards RY repeat which can adopt unusual DNA conformation, we have investigated the effects of the superhelicity-dependent conformational transition of the 52 bp RY repeat on topoisomerase II cleavages. Cleavage reactions were performed on the pRYG plasmid with varying levels of negative superhelical densities ranging from 0 to -0.074. Under the low torsional stress, topoisomerase II cleavage activity at the RY repeat gradually increased with the increasing levels of negative superhelical densities. However, over a threshold level of negative supercoiling for cruciform conformation, the intensities of enzyme cleavage sites at the RY repeat were essentially identical. These results suggest that topoisomerase II can bind and cleave the cruciform structure in a dynamic process identical to duplex B-DNA.