Positioning the 3′-DNA Terminus for Topoisomerase II-mediated Religation

Despite the importance of the topoisomerase II DNA cleavage/rejoining cycle to genomic integrity, the mechanistic details of religation are poorly understood. Topoisomerase II utilizes covalent protein-DNA interactions to align the 5′-termini of cleaved DNA for religation. However, because the enzyme does not form covalent bonds with the 3′-DNA termini, the basis for the alignment of the 3′-ends is less clear. Three major possibilities exist. The 3′-termini may be positioned for religation (i) by base pairing to their complementary DNA strands, (ii) by base stacking to the adjacent residues, or (iii) by noncovalent interactions with topoisomerase II. To distinguish between these possibilities, the ability of human topoisomerase IIα to religate a series of oligonucleotides with altered base pairing or base stacking at their 3′-termini was determined. Substrates containing modifications that disrupted terminal base pairing or base stacking with-out affecting the 3′-terminal base were resealed at wild-type rates. In contrast, substrates that lacked the terminal base (or contained an altered base) displayed very low rates of religation. On the basis of these results, we propose that topoisomerase II positions the 3′-DNA termini for religation through noncovalent protein-DNA contacts.