We propose to continue our work to investigate deeper into the mechanism of activation of a local origin gamma by initiator protein pi-ori interaction, activation of a distant ori by DNA looping, inactivation of an ori by handcuffing, in the plasmid R6K. We propose to use a newly reconstituted replication system consisting of 22 purified proteins to investigate the detailed mechanism of activation of the local origin gamma. We propose to identify the combination of chaperones (and any other proteins) needed to activate the dimeric and inert WT p so that it not only activates the local origin gamma, but is also able to activate the distant origins alpha and beta by DNA looping in vitro. The crystal structure of the monomeric initiator pi* bound to a single iteron DNA will be solved and the structure used to gain additional insights into the mechanism of initiation of replication at atomic resolution. Our long term goal is to extend crystallography further by co-crystallizing ternary complexes of peptides of DnaB, DnaA, DnaG that interact with p -iteron DNA complex with the objective of further extending our knowledge of replication initiation at high resolution.