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 are outlined in this proposal. Building on the results of Alberts and Nossal and their co- workers, who have defined this in vitro system, as well as on our own earlier work, we will study the mechanism of action of each functional subset of proteins, and how these subsets are assembled into the integrated DNA replication complex. These subsets include the central DNA polymerase (gene 43 protein), the Polymerase accessory proteins complex (genes 44/62 and 45 proteins), and the helicase-primase complex (genes 41 and 61 proteins), each of which also interacts with the T4 single-stranded DNA binding (gene 32) protein. During the next granting period we will use a number of enzymatic and biophysical methods to """"""""probe"""""""" the structure, assembly, and function of the accessory proteins complex, the helicase- primase complex, the five-protein holoenzyme complex (polymerase plus accessory proteins), and the integrated seven-protein system (holoenzyme plus helicase-primase). Methods to be used include UV laser protein-DNA crosslinking, rapid-quench kinetics, and cryoelectron microscopy, as well as the usual physical biochemical and enzymatic techniques. The processivity (as well as the fidelity) of the polymerase will be used 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). We will continue to develop a molecular and a kinetic model of the individual steps of the processive single-nucleotide addition (or excision) cycle of the polymerase, to determine which of these steps are regulated by the accessory proteins complex, and to ask how the functions of paired holoenzyme complexes are integrated structurally, mechanistically, and via the helicase-primase complex. We hope that these studies will contribute to a further molecular understanding of the various sets of protein-protein and protein-nucleic acid interactions that control the function of this DNA elongation complex, as well as further insights into how the complex may be regulated in various physiological states.
Showing the most recent 10 out of 112 publications
