TOPOISOMERASE II-MEDIATED DNA CLEAVAGE AND RELIGATION IN THE ABSENCE OF BASE-PAIRING - ABASIC LESIONS AS A TOOL TO DISSECT ENZYME MECHANISM

The interaction of topoisomerase II with its DNA cleavage site is crit ical to the physiological functions of the enzyme. Despite this import ance, the specific enzyme-DNA interactions that drive topoisomerase II -mediated DNA cleavage and religation are poorly understood. Therefore , to dissect interactions between the enzyme and its cleavage site, ab asic DNA lesions were incorporated into a bilaterally symmetrical and identical cleavage site, Results indicate that topoisomerase II has un ique interactions with each position of the 4-base overhang generated by enzyme-mediated DNA cleavage. Lesions located 2 bases 3' to the poi nt of scission stimulated cleavage the most, whereas those 3 bases fro m the point of scission stimulated cleavage the least. Moreover, an ad ditive and in some cases synergistic cleavage enhancement was observed in oligonucleotides that contained multiple DNA lesions, with levels reaching >60-fold higher than the wild-type substrate. Finally, topois omerase II efficiently cleaved and religated a DNA substrate in which apyrimidinic sites were simultaneously incorporated at every position on one strand of the 4-base overhang. Therefore, unlike classical DNA ligases in which base pairing is the driving force behind closure of t he DNA break, it appears that for topoisomerase II, the enzyme is resp onsible for the spatial orientation of the DNA termini for ligation.