The long range goal of the proposed studies is to define the fundamental molecular mechanisms that underpin initiation and regulation of bacteriophage ? DNA replication in a model in vitro system composed of 20 or more highly purified ? and E. coli replication proteins. The proposed studies are expected to provide insights that will guide studies of the replication of chromosomes of more complex organisms, including all eubacteria and many human DNA tumor viruses. The ? O and P replication initiators promote the assembly of a pre-replicative complex (pre-RC) at the phage replication origin (ori?) that contains the 2 initiators and the E. coli DnaB replicative helicase. A critical stage in the initiation pathway is the conversion of this functionally inert nucleoprotein complex into an """"""""activated pre-RC"""""""" upon negative supercoiling of the DNA template. We will use a variety of biochemical approaches, including the use of photoaffinity protein-DNA crosslinking or mutant initiator proteins, to define the nature of the protein-DNA interactions in this key replication intermediate. We will seek to confirm our hypothesis that cryptic single-stranded-DNA (ssDNA) binding activities associated with the ?, O and P initiator proteins are responsible for stabilizing a conformational variant of the DNA duplex at ori?, to activate the DNA for loading of DnaB helicase. Related approaches will be used to precisely define the pathway by which a ring-shaped DnaB hexamer is transferred onto the ? chromosome at the Arich region of ori?, during nucleoprotein remodeling catalyzed by E. coli molecular chaperones. We will pursue crystallographic studies to determine the three-dimensional (3D) structure of the DNA-binding domain of O, bound to its recognition site. A related aim is to elucidate the structure of the ? pre-RC assembled on forked ori? DNA templates or on origin DNA that contains small bubbles or mismatches. Biochemical and genetic studies will be performed to define the ssDNA-binding motifs in ? P and the C-terminal domain of O. We will determine the molecular mechanism by which transcription of the DNA template in cis activates ? DNA replication.
