Double-strand cleavage and strand joining by the replication initiator protein of filamentous phage f1

The replication initiator protein (gene II protein (gpII)) of bacteriophage f1 is a multifunctional protein that plays central roles in initiation and termination of phage DNA replication. It introduces a nick at a specific site on the (+)-strand of supercoiled replicative form DNA. The 3'-hydroxyl end of the nick serves as the primer for (+)-strand rolling-circle replication. Upon completion of a round of synthesis, gpII cleaves and circularizes the displaced single strand. When Mn²⁺ is included in the buffer instead of Mg²⁺, gpII cleaves both strands. In this paper, we investigate the mechanism of the Mn²⁺-dependent double-strand cleavage activity of gpII. This reaction, unlike nicking in the presence of Mg²⁺, does not require superhelicity. The reaction proceeds in two kinetic steps: first nicking of the (+)-strand, and then cleavage of the (-)-strand. The nucleotide sequence requirement for nicking is reduced compared to that in the presence of Mg²⁺. The product of the double-strand cleavage has an unusual structure. The left end is a telomere-like hairpin since the (+)- and (-)-strands are joined, as demonstrated by base sequencing. The right end has a one-base 3'-overhang. This reaction probably reflects the cleavage-joining activity of gpII in the termination event.