Protelomerase Uses a Topoisomerase IB/ Y-Recombinase Type Mechanism to Generate DNA Hairpin Ends

Protelomerases are enzymes responsible for the generation of closed hair-pin ends in linear DNA. It is proposed that they use a breaking-and-rejoin type mechanism to affect DNA rearrangement on specific DNA sequences. In doing so, one strand turns around and becomes the comp-lementary strand. Using the purified enzyme from the Escherichia coli phage N15 and the Klebsiella phage fKO2 and synthetic oligonucleotide substrates, we directly demonstrate the location where the cutting/re-lig-ation occurs. We identified a pair of transient staggered cleavages six base-pairs apart centered around the axis of dyad symmetry of the target site. Two molecules of the protelomerase form a pair of protein-linked DNA intermediates at each 30 end of the cleaved openings leaving a 50-OH. Then, in a process not yet clearly defined, the partners of the two initial openings are exchanged, and the transient breaks are resealed to generate hairpin ends. The formation of 30-covalent DNA–protein inter-mediates is a hallmark of the topoisomerase IB type reaction, and w