Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where 35 S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.Many phages depend on the host DNA replication machinery, but they usually code for one or two proteins that direct the host machinery to their own genomes. For example, phage codes for O, an origin-binding protein, and P, a protein that interacts with O as well as with Escherichia coli DnaB helicase (36). The P protein can thus direct the helicase to the phage origin and is considered an analogue of E. coli DnaC protein, which loads the helicase onto the bacterial origin (51).Bacteriophage P2 is a temperate coliphage that replicates via a modified rolling-circle mechanism generating monomeric double-stranded circles (7). P2 also utilizes several host components for its replication, like DNA polymerase III, primase, DnaB, and the Rep helicase (9, 11), but encodes at least two proteins for its own replication. One is protein A, which initiates replication by introducing a sequence-specific, singlestranded cut at the origin of replication (ori) (33). The second P2 protein required for phage replication is the B protein (31,32). It is believed to be required for lagging-strand synthesis, since the displaced strand during rolling-circle replication remains single stranded in the absence of B (20). Since laggingstrand synthesis requires loading of the helicase (DnaB)-primase (DnaG) complex, a defect in helicase loading could account for the result. The requirement for the E. coli DnaB protein in P2 DNA replication is known (9). Moreover, a P2 rlb1 mutation, within the coding part of the B gene, has been shown to partially suppress a dnaB(Ts) mutation (49), suggesting that P2 B interacts directly with DnaB. These results led to the hypothesis that it is a DnaC analogue (23). However, the B protein was not required for replication of a P2 minichromosome containing only the P2 A gene...