We propose to continue our investigation of mechanisms by which viruses seize the replication machinery of their host cell and direct this machinery to function solely on the invading viral chromosome. The bacteriophage Lambda - Escherichia coli interaction has been chosen as a model system for study of this problem. This choice is based upon the availability of all viral and host replication proteins in a purified form, as well as upon the development in this laboratory of an in vitro DNA replication system in which these proteins act. This system should be highly amenable to characterization of the functional roles and molecular interactions of the viral and bacterial replication proteins. The bacteriophage Lambda O and P replication proteins cooperate with six E. coli proteins to catalyze the initiation of complementary strands on single-stranded circular viral DNAs. An activated nucleoprotein complex containing the bacterial dnaB protein is assembled in an ATP-dependent reaction prior to priming and DNA synthesis. We will attempt to define the pathway of assembly of this critical nucleoprotein prepriming complex and to elucidate the molecular mechanisms of the proteins that function in this reaction. This study will involve the isolation and characterization of nucleoprotein complexes that are precursors to the complex that is ultimately generated. Identification and quantitation of the proteins present in each structure will depend on the use of radioisotopically labeled proteins or on immunoblotting techniques that utilize antibodies directed against specific replication proteins. We will continue to define the physical and functional properties of those host and virus replication proteins that participate in the assembly of the activated prepriming complex. We will continue to study the specific interaction between the Lambda P protein and the bacterial dnaB protein and to contrast this interaction with the analogous interaction between the dnaB and dnaC host proteins. We will investigate the effect of the E. coli dnaJ and dnaK proteins on the stability of the Lambda P-dnaB protein complex. We will examine the nature of the interaction between the Lambda O and P initiator proteins. We will characterize the functional properties of the bacterial dnaJ and dnaK proteins in a single-strand DNA replication system. Particular emphasis will be placed on studies of the autophosphorylation of dnaK protein and the role of this reaction in the assembly of nucleoprotein prepriming complexes.
Showing the most recent 10 out of 11 publications
