Chromosomes must be duplicated for cells to divide. Chromosomal replication is carried out by a multiprotein machine. The chromosomal duplicating machine of E. coil is called DNA polymerase III holoenzyme. The holoenzyme is composed of 10 different proteins, some in a stoichiometry of two or more, for a total of 18 polypeptide chains. Within this machine are two DNA polymerases for coordinated synthesis of both strands of duplex DNA. The holoenzyme machinery also contains two beta sliding clamps which encircle DNA and act as a mobile tether to hold the machinery down to DNA for highly processive chromosome duplication. The holoenzyme also contains a 5-subunit "clamp loader" which acts as a protein topoisomerase to couple ATP hydrolysis to assemble the beta clamp around DNA. This proposal seeks a greater understanding of how the DNA polymerase III holoenzyme acts during chromosome duplication. The mechanism of the clamp loader in opening up beta rings and the use of ATP in this process will be studied in molecular detail. The semidiscontinuous mode of replication requires the polymerase on the lagging strand to cycle on and off DNA, one time for each fragment. Hence, polymerase comes on and off DNA every second or two, even though it is held tightly to DNA by a beta ring. The mechanism entails polymerase dissociation from the bring upon recognition that a DNA fragment is complete, and rapid reassociation with a new beta ring. How the polymerase on the lagging strand performs this task, the subunits involved in the process, and use of ATP will be investigated. Also, beta clamps that remain on DNA as the polymerase hops from one fragment to the other must be recycled. The mechanism by which beta clamps are removed from DNA for use on future Okazaki fragments will be investigated. The replication machine also acts with the helicase that unwinds the parental duplex, and with primase which synthesizes RNA primers for the discontinuous lagging strand. How this "replisome" machinery deals with proteins bound to DNA, and DNA lesions, will be studied. Finally, the replicating machine must assemble at an origin to form replication_forks. The process by which replisomes assemble into the origin of E. coli will be studied.