The long term goal of my laboratory is to understand the relationship between the architecture and function of the proteins that participate in bacteriophage T4 DNA replication. The choice of T4 phage DNA replication was based on the extensive genetic, and biochemical and biophysical information that has already been amassed on this system, the fact that it can be readily studied in vitro, and the belief that it provides a reasonable model for DNA replication in higher organisms. Knowledge of the structure of the proteins involved in T4 DNA replication as well as the organization and regulation of the relevant genes is required before a detailed understanding of the functionally significant protein-protein and protein-DNA interactions can be achieved. Accordingly we have determined the nucleotide sequence of several of the structural genes coding for the T4 DNA replication proteins and we have identified the control regions that regulate their expression. We are attempting to clone the gene for T4 DNA polymerase and the other replication accessory protein genes in controllable, high expression vectors with the aim of obtaining enough of the proteins to carry out structure-function studies. These will include crystallization selective in vitro mutagenesis, limited proteolysis to look for separate functional domains, and physico-chemical studies that should illuminate the nature of there interaction with each other and with DNA. We will try to understand the mechanism of T4 DNA polymerase autoregulation as well. Studies on the T4 single strand DNA binding protein (32P), an essential component of the replication complex, and other single strand DNA binding proteins from both prokaryotes and eukaryotes are continuing with emphasis on physical-chemical studies that should show which residues are involved in binding DNA and which regions are necessary for interaction with other proteins in the replication complex. Attempts will be made to clone three of the T4 genes, uvsX, Y and W whose products appear to be involved in the """"""""late"""""""" mode of T4 DNA replication. Assuming that we can obtain a high level of expression of the uvsX, Y and W proteins, we will examine their properties with particular emphasis on the uvsX protein which appears to behave in a fashion similar the recA protein from E. coli.
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