Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein

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Abstract
Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an Initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut Induced by the A protein In vivo is determined In this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (Ml) primer extensions. It Is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, In a region devold of secondary structures. The A gene has been cloned Into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but It cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.