Purified bacteriophage λ O protein binds to four repeating sequences at the λ replication origin

Abstract

The bacteriophage lambda O protein is needed for initiation of lambda DNA replication. Several lines of evidence suggest that initiation requires that this protein interacts with a specific sequence called ori (for origin) in lambda DNA. We have purified this protein to near homogeneity and studied the protection against nuclease cleavage of the origin DNA sequences. Our data demonstrate that the O protein binds within an interval of about 95 base pairs (bp), which contains four tandemly arranged 19bp repeating sequences, ATCCCTCAAAACGA (G)GG GAT(A). At a low concentration of O protein, the inner two repeats are primarily covered, while binding to the outer two repeats requires a high concentration of O protein. From the molecular size of O protein (32,000 daltons), and the internal symmetry in each 19bp repeat, we inferred that the O protein may bind in dimeric form, and that the 95bp region may be filled only when four such dimers have bound. This interaction is discussed in connection with the "activation" of the ori by O protein leading to initiation of DNA synthesis.