In this proposal we outline the next stages of a continuing physical biochemical study of the structural and functional interactions of the protein and nucleic acid components of the bacteriophage T4 DNA replication system. Building on the results of Alberts and Nossal and their coworkers, who have defined this in vitro system, as well as on our own earlier studies, we will continue to focus on a step-wise """"""""building-up"""""""" of the complex to gain further insight into the functional role of each component in the integrated system. To this end we plan to continue to use our newly developed UV laser cross-linking methodology to determine contacts (and affinities) between the various proteins of the system and the DNA of the primer-template junction, as well as the DNA strand displaced by the advancing replication fork in asymmetric (leading-strand-only) DNA synthesis. We will also continue to study the assembly and molecular activities of functional subsets of the replication system, using enzymatic and physical chemical """"""""probes"""""""". These subsets include the DNA polymerase (gene 43 protein) itself, the polymerase accessory-helicase complex (genes 44/62 + 45 proteins) and the lagging strand primase-helicase complex (genes 41 and 61 protein), each of which also interacts with the T4 single-strand DNA binding (gene 32) protein. We will also continue to study the processivity of the polymerase as a measure of the functional integration of the various components of the system (working both """"""""forward"""""""" in DNA synthesis and """"""""backward"""""""" as an exonuclease """"""""editor"""""""" of the growing primer strand), and plan to use this approach to begin to study the issue of the fidelity of DNA synthesis as well. We hope that these studies will help to provide at least a partial molecular understanding of the various sets of protein-protein and protein-nucleic acid interactions that control the function of this DNA elongation complex, and of how these interactions work together to form a coupled system that conduct leading- and lagging-strand DNA synthesis at physiological rates and with physiological fidelities.
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